Bispecific t cell activating antigen binding molecules

ABSTRACT

The present invention generally relates to novel bispecific antigen binding molecules for T cell activation and re-direction to specific target cells. In addition, the present invention relates to polynucleotides encoding such bispecific antigen binding molecules, and vectors and host cells comprising such polynucleotides. The invention further relates to methods for producing the bispecific antigen binding molecules of the invention, and to methods of using these bispecific antigen binding molecules in the treatment of disease.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of Ser. No. 17/711,489 filed Apr. 1,2022, which is a divisional of Ser. No. 17/400,998 filed Aug. 12, 2021,now abandoned, which is a divisional of Ser. No. 16/799,662 filed Feb.24, 2020, now U.S. Pat. No. 11,117,965, which is a divisional of U.S.patent application Ser. No. 15/879,040, filed Jan. 24, 2018, now U.S.Pat. No. 10,611,840, which is a divisional of U.S. patent applicationSer. No. 14/816,252, filed Aug. 3, 2015, now U.S. Pat. No. 9,914,776,which claims priority to European Patent Application No. EP 15170866.6,filed Jun. 5, 2015, and to European Patent Application No. EP14179764.7, filed Aug. 4, 2014, the disclosures of which areincorporated herein by reference in their entirety.

SEQUENCE LISTING

The present application contains a Sequence Listing which has beensubmitted in XML format and is hereby incorporated by reference in itsentirety. Said XML copy, created on Oct. 6, 2022, is named51177-009007_Sequence_Listing_10_6_22_ST26 and is 97,487 bytes in size.

FIELD OF THE INVENTION

The present invention generally relates to bispecific antigen bindingmolecules for activating T cells. In addition, the present inventionrelates to polynucleotides encoding such bispecific antigen bindingmolecules, and vectors and host cells comprising such polynucleotides.The invention further relates to methods for producing the bispecificantigen binding molecules of the invention, and to methods of usingthese bispecific antigen binding molecules in the treatment of disease.

BACKGROUND

The selective destruction of an individual cell or a specific cell typeis often desirable in a variety of clinical settings. For example, it isa primary goal of cancer therapy to specifically destroy tumor cells,while leaving healthy cells and tissues intact and undamaged.

An attractive way of achieving this is by inducing an immune responseagainst the tumor, to make immune effector cells such as natural killer(NK) cells or cytotoxic T lymphocytes (CTLs) attack and destroy tumorcells. CTLs constitute the most potent effector cells of the immunesystem, however they cannot be activated by the effector mechanismmediated by the Fc domain of conventional therapeutic antibodies.

In this regard, bispecific antibodies designed to bind with one “arm” toa surface antigen on target cells, and with the second “arm” to anactivating, invariant component of the T cell receptor (TCR) complex,have become of interest in recent years. The simultaneous binding ofsuch an antibody to both of its targets will force a temporaryinteraction between target cell and T cell, causing activation of anycytotoxic T cell and subsequent lysis of the target cell. Hence, theimmune response is re-directed to the target cells and is independent ofpeptide antigen presentation by the target cell or the specificity ofthe T cell as would be relevant for normal MHC-restricted activation ofCTLs. In this context it is crucial that CTLs are only activated when atarget cell is presenting the bispecific antibody to them, i.e. theimmunological synapse is mimicked. Particularly desirable are bispecificantibodies that do not require lymphocyte preconditioning orco-stimulation in order to elicit efficient lysis of target cells.

Several bispecific antibody formats have been developed and theirsuitability for T cell mediated immunotherapy investigated. Out ofthese, the so-called BiTE (bispecific T cell engager) molecules havebeen very well characterized and already shown some promise in theclinic (reviewed in Nagorsen and Bäuerle, Exp Cell Res 317, 1255-1260(2011)). BiTEs are tandem scFv molecules wherein two scFv molecules arefused by a flexible linker. Further bispecific formats being evaluatedfor T cell engagement include diabodies (Holliger et al., Prot Eng 9,299-305 (1996)) and derivatives thereof, such as tandem diabodies(Kipriyanov et al., J Mol Biol 293, 41-66 (1999)). A more recentdevelopment are the so-called DART (dual affinity retargeting)molecules, which are based on the diabody format but feature aC-terminal disulfide bridge for additional stabilization (Moore et al.,Blood 117, 4542-51 (2011)). The so-called triomabs, which are wholehybrid mouse/rat IgG molecules and also currently being evaluated inclinical trials, represent a larger sized format (reviewed in Seimetz etal., Cancer Treat Rev 36, 458-467 (2010)).

The variety of formats that are being developed shows the greatpotential attributed to T cell re-direction and activation inimmunotherapy. The task of generating bispecific antibodies suitabletherefor is, however, by no means trivial, but involves a number ofchallenges that have to be met related to efficacy, toxicity,applicability and producibility of the antibodies.

Small constructs such as, for example, BiTE molecules—while being ableto efficiently crosslink effector and target cells—have a very shortserum half life requiring them to be administered to patients bycontinuous infusion. IgG-like formats on the other hand—while having thegreat benefit of a long half life—suffer from toxicity associated withthe native effector functions inherent to IgG molecules. Theirimmunogenic potential constitutes another unfavorable feature ofIgG-like bispecific antibodies, especially non-human formats, forsuccessful therapeutic development. Finally, a major challenge in thegeneral development of bispecific antibodies has been the production ofbispecific antibody constructs at a clinically sufficient quantity andpurity, due to the mispairing of antibody heavy and light chains ofdifferent specificities upon co-expression, which decreases the yield ofthe correctly assembled construct and results in a number ofnon-functional side products from which the desired bispecific antibodymay be difficult to separate.

Different approaches have been taken to overcome the chain associationissue in bispecific antibodies (see e.g. Klein et al., mAbs 6, 653-663(2012)). For example, the ‘knobs-into-holes’ strategy aims at forcingthe pairing of two different antibody heavy chains by introducingmutations into the CH3 domains to modify the contact interface. On onechain bulky amino acids are replaced by amino acids with short sidechains to create a ‘hole’. Conversely, amino acids with large sidechains are introduced into the other CH3 domain, to create a ‘knob’. Bycoexpressing these two heavy chains (and two identical light chains,which have to be appropriate for both heavy chains), high yields ofheterodimer (‘knob-hole’) versus homodimer (‘hole-hole’ or ‘knob-knob’)are observed (Ridgway, J. B., et al., Protein Eng. 9 (1996) 617-621; andWO 96/027011). The percentage of heterodimer could be further increasedby remodeling the interaction surfaces of the two CH3 domains using aphage display approach and the introduction of a disulfide bridge tostabilize the heterodimers (Merchant, A. M., et al., Nature Biotech. 16(1998) 677-681; Atwell, S., et al., J. Mol. Biol. 270 (1997) 26-35). Newapproaches for the knobs-into-holes technology are described in e.g. inEP 1870459 A1.

The ‘knobs-into-holes’ strategy does, however, not solve the problem ofheavy chain-light chain mispairing, which occurs in bispecificantibodies comprising different light chains for binding to thedifferent target antigens.

A strategy to prevent heavy chain-light chain mispairing is to exchangedomains between the heavy and light chains of one of the binding arms ofa bispecific antibody (see WO 2009/080251, WO 2009/080252, WO2009/080253, WO 2009/080254 and Schaefer, W. et al, PNAS, 108 (2011)11187-11191, which relate to bispecific IgG antibodies with a domaincrossover).

Exchanging the heavy and light chain variable domains VH and VL in oneof the binding arms of the bispecific antibody (WO2009/080252, see alsoSchaefer, W. et al, PNAS, 108 (2011) 11187-11191) clearly reduces theside products caused by the mispairing of a light chain against a firstantigen with the wrong heavy chain against the second antigen (comparedto approaches without such domain exchange). Nevertheless, theseantibody preparations are not completely free of side products. The mainside product is based on a Bence Jones-type interaction (Schaefer, W. etal, PNAS, 108 (2011) 11187-11191; in Fig. S1I of the Supplement). Afurther reduction of such side products is thus desirable to improvee.g. the yield of such bispecific antibodies.

Given the difficulties and disadvantages associated with currentlyavailable bispecific antibodies for T cell mediated immunotherapy, thereremains a need for novel, improved formats of such molecules. Thepresent invention provides bispecific antigen binding molecules designedfor T cell activation and re-direction that combine good efficacy andproduceability with low toxicity and favorable pharmacokineticproperties.

SUMMARY OF THE INVENTION

According to the invention, the ratio of a desired bispecific antibodycompared to undesired side products, in particular Bence Jones-type sideproducts occurring in bispecific antibodies with a VH/VL domain exchangein one of their binding arms, can be improved by the introduction ofcharged amino acids with opposite charges at specific amino acidpositions in the CH1 and CL domains.

Thus, in a first aspect the present invention provides a T cellactivating bispecific antigen binding molecule comprising

-   -   (a) a first Fab molecule which specifically binds to a first        antigen;    -   (b) a second Fab molecule which specifically binds to a second        antigen, and wherein the variable domains VL and VH of the Fab        light chain and the Fab heavy chain are replaced by each other;

wherein the first antigen is an activating T cell antigen and the secondantigen is a target cell antigen, or the first antigen is a target cellantigen and the second antigen is an activating T cell antigen; andwherein

-   -   i) in the constant domain CL of the first Fab molecule under a)        the amino acid at position 124 is substituted independently by        lysine (K), arginine (R) or histidine (H) (numbering according        to Kabat), and wherein in the constant domain CH1 of the first        Fab molecule under a) the amino acid at position 147 or the        amino acid at position 213 is substituted independently by        glutamic acid (E), or aspartic acid (D) (numbering according to        Kabat EU index); or    -   ii) in the constant domain CL of the second Fab molecule        under b) the amino acid at position 124 is substituted        independently by lysine (K), arginine (R) or histidine (H)        (numbering according to Kabat), and wherein in the constant        domain CH1 of the second Fab molecule under b) the amino acid at        position 147 or the amino acid at position 213 is substituted        independently by glutamic acid (E), or aspartic acid (D)        (numbering according to Kabat EU index).

According to the invention, the second Fab molecule is a crossover Fabmolecule wherein the variable regions of the Fab light chain and the Fabheavy chain are exchanged. In particular embodiments, the first (and thethird, if any) Fab molecule is a conventional Fab molecule. In a furtherparticular embodiment, not more than one Fab molecule capable ofspecific binding to an activating T cell antigen is present in the Tcell activating bispecific antigen binding molecule (i.e. the T cellactivating bispecific antigen binding molecule provides monovalentbinding to the activating T cell antigen).

In a particular embodiment, the first antigen is a target cell antigenand the second antigen is an activating T cell antigen. In a morespecific embodiment, the activating T cell antigen is CD3, particularlyCD3 epsilon. In one embodiment, the target cell antigen is CD20.

In one embodiment of the T cell activating bispecific antigen bindingmolecule according to the invention, in the constant domain CL of thefirst Fab molecule under a) the amino acid at position 124 issubstituted independently by lysine (K), arginine (R) or histidine (H)(numbering according to Kabat) (in one preferred embodimentindependently by lysine (K) or arginine (R)), and in the constant domainCH1 of the first Fab molecule under a) the amino acid at position 147 orthe amino acid at position 213 is substituted independently by glutamicacid (E), or aspartic acid (D) (numbering according to Kabat EU index).

In a further embodiment, in the constant domain CL of the first Fabmolecule under a) the amino acid at position 124 is substitutedindependently by lysine (K), arginine (R) or histidine (H) (numberingaccording to Kabat), and in the constant domain CH1 of the first Fabmolecule under a) the amino acid at position 147 is substitutedindependently by glutamic acid (E), or aspartic acid (D) (numberingaccording to Kabat EU index).

In yet another embodiment, in the constant domain CL of the first Fabmolecule under a) the amino acid at position 124 is substitutedindependently by lysine (K), arginine (R) or histidine (H) (numberingaccording to Kabat) (in one preferred embodiment independently by lysine(K) or arginine (R)) and the amino acid at position 123 is substitutedindependently by lysine (K), arginine (R) or histidine (H) (numberingaccording to Kabat) (in one preferred embodiment independently by lysine(K) or arginine (R)), and in the constant domain CH1 of the first Fabmolecule under a) the amino acid at position 147 is substitutedindependently by glutamic acid (E), or aspartic acid (D) (numberingaccording to Kabat EU index) and the amino acid at position 213 issubstituted independently by glutamic acid (E), or aspartic acid (D)(numbering according to Kabat EU index).

In a particular embodiment, in the constant domain CL of the first Fabmolecule under a) the amino acid at position 124 is substituted bylysine (K) (numbering according to Kabat) and the amino acid at position123 is substituted by lysine (K) (numbering according to Kabat), and inthe constant domain CH1 of the first Fab molecule under a) the aminoacid at position 147 is substituted by glutamic acid (E) (numberingaccording to Kabat EU index) and the amino acid at position 213 issubstituted by glutamic acid (E) (numbering according to Kabat EUindex).

In another particular embodiment, in the constant domain CL of the firstFab molecule under a) the amino acid at position 124 is substituted bylysine (K) (numbering according to Kabat) and the amino acid at position123 is substituted by arginine (R) (numbering according to Kabat), andin the constant domain CH1 of the first Fab molecule under a) the aminoacid at position 147 is substituted by glutamic acid (E) (numberingaccording to Kabat EU index) and the amino acid at position 213 issubstituted by glutamic acid (E) (numbering according to Kabat EUindex).

In one embodiment, the T cell activating bispecific antigen bindingmolecule of the invention comprises

-   -   (a) a first Fab molecule which specifically binds to a first        antigen;    -   (b) a second Fab molecule which specifically binds to a second        antigen, and wherein the variable domains VL and VH of the Fab        light chain and the Fab heavy chain are replaced by each other;

wherein the first antigen is a target cell antigen and the secondantigen is an activating T cell antigen; and

wherein in the constant domain CL of the first Fab molecule under a) theamino acid at position 124 is substituted independently by lysine (K),arginine (R) or histidine (H) (numbering according to Kabat) (in onepreferred embodiment independently by lysine (K) or arginine (R)) andthe amino acid at position 123 is substituted independently by lysine(K), arginine (R) or histidine (H) (numbering according to Kabat) (inone preferred embodiment independently by lysine (K) or arginine (R)),and in the constant domain CH1 of the first Fab molecule under a) theamino acid at position 147 is substituted independently by glutamic acid(E), or aspartic acid (D) (numbering according to Kabat EU index) andthe amino acid at position 213 is substituted independently by glutamicacid (E), or aspartic acid (D) (numbering according to Kabat EU index).In an alternative embodiment of the T cell activating bispecific antigenbinding molecule according to the invention, in the constant domain CLof the second Fab molecule under b) the amino acid at position 124 issubstituted independently by lysine (K), arginine (R) or histidine (H)(numbering according to Kabat) (in one preferred embodimentindependently by lysine (K) or arginine (R)), and in the constant domainCH1 of the second Fab molecule under b) the amino acid at position 147or the amino acid at position 213 is substituted independently byglutamic acid (E), or aspartic acid (D) (numbering according to Kabat EUindex).

In a further embodiment, in the constant domain CL of the second Fabmolecule under b) the amino acid at position 124 is substitutedindependently by lysine (K), arginine (R) or histidine (H) (numberingaccording to Kabat), and in the constant domain CH1 of the second Fabmolecule under b) the amino acid at position 147 is substitutedindependently by glutamic acid (E), or aspartic acid (D) (numberingaccording to Kabat EU index).

In still another embodiment, in the constant domain CL of the second Fabmolecule under b) the amino acid at position 124 is substitutedindependently by lysine (K), arginine (R) or histidine (H) (numberingaccording to Kabat) (in one preferred embodiment independently by lysine(K) or arginine (R)) and the amino acid at position 123 is substitutedindependently by lysine (K), arginine (R) or histidine (H) (numberingaccording to Kabat) (in one preferred embodiment independently by lysine(K) or arginine (R)), and in the constant domain CH1 of the second Fabmolecule under b) the amino acid at position 147 is substitutedindependently by glutamic acid (E), or aspartic acid (D) (numberingaccording to Kabat EU index) and the amino acid at position 213 issubstituted independently by glutamic acid (E), or aspartic acid (D)(numbering according to Kabat EU index).

In one embodiment, in the constant domain CL of the second Fab moleculeunder b) the amino acid at position 124 is substituted by lysine (K)(numbering according to Kabat) and the amino acid at position 123 issubstituted by lysine (K) (numbering according to Kabat), and in theconstant domain CH1 of the second Fab molecule under b) the amino acidat position 147 is substituted by glutamic acid (E) (numbering accordingto Kabat EU index) and the amino acid at position 213 is substituted byglutamic acid (E) (numbering according to Kabat EU index).

In another embodiment, in the constant domain CL of the second Fabmolecule under b) the amino acid at position 124 is substituted bylysine (K) (numbering according to Kabat) and the amino acid at position123 is substituted by arginine (R) (numbering according to Kabat), andin the constant domain CH1 of the second Fab molecule under b) the aminoacid at position 147 is substituted by glutamic acid (E) (numberingaccording to Kabat EU index) and the amino acid at position 213 issubstituted by glutamic acid (E) (numbering according to Kabat EUindex).

In some embodiments, the T cell activating bispecific antigen bindingmolecule according to the invention further comprises a third Fabmolecule which specifically binds to the first antigen.

In particular embodiments, the third Fab molecule is identical to thefirst Fab molecule. In these embodiments, the third Fab molecule thuscomprises the same amino acid substitutions as the first Fab molecule.Like the first Fab molecule, the third Fab molecule particularly is aconventional Fab molecule.

If a third Fab molecule is present, in a particular embodiment the firstand the third Fab molecule specifically bind to a target cell antigen,and the second Fab molecule specifically binds to an activating T cellantigen, particularly CD3, more particularly CD3 epsilon.

In some embodiments of the T cell activating bispecific antigen bindingmolecule according to the invention the first Fab molecule under a) andthe second Fab molecule under b) are fused to each other, optionally viaa peptide linker. In a specific embodiment, the second Fab molecule isfused at the C-terminus of the Fab heavy chain to the N-terminus of theFab heavy chain of the first Fab molecule.

In an alternative embodiment, the first Fab molecule is fused at theC-terminus of the Fab heavy chain to the N-terminus of the Fab heavychain of the second Fab molecule. In embodiments wherein either (i) thesecond Fab molecule is fused at the C-terminus of the Fab heavy chain tothe N-terminus of the Fab heavy chain of the first Fab molecule or (ii)the first Fab molecule is fused at the C-terminus of the Fab heavy chainto the N-terminus of the Fab heavy chain of the second Fab molecule,additionally the Fab light chain of the Fab molecule and the Fab lightchain of the second Fab molecule may be fused to each other, optionallyvia a peptide linker.

In particular embodiments, the T cell activating bispecific antigenbinding molecule according to the invention additionally comprises an Fcdomain composed of a first and a second subunit capable of stableassociation.

The T cell activating bispecific antigen binding molecule according tothe invention can have different configurations, i.e. the first, second(and optionally third) Fab molecule may be fused to each other and tothe Fc domain in different ways. The components may be fused to eachother directly or, preferably, via one or more suitable peptide linkers.Where fusion is to the N-terminus of a subunit of the Fc domain, it istypically via an immunoglobulin hinge region.

In one embodiment, the second Fab molecule is fused at the C-terminus ofthe Fab heavy chain to the N-terminus of the first or the second subunitof the Fc domain. In such embodiment, the first Fab molecule may befused at the C-terminus of the Fab heavy chain to the N-terminus of theFab heavy chain of the second Fab molecule or to the N-terminus of theother one of the subunits of the Fc domain.

In one embodiment, the first and the second Fab molecule are each fusedat the C-terminus of the Fab heavy chain to the N-terminus of one of thesubunits of the Fc domain. In this embodiment, the T cell activatingbispecific antigen binding molecule essentially comprises animmunoglobulin molecule, wherein in one of the Fab arms the heavy andlight chain variable regions VH and VL are exchanged/replaced by eachother (see FIGS. 1A and 1D).

In alternative embodiments, the third Fab molecule is fused at theC-terminus of the Fab heavy chain to the N-terminus of the first orsecond subunit of the Fc domain. In a particular such embodiment, thesecond and the third Fab molecule are each fused at the C-terminus ofthe Fab heavy chain to the N-terminus of one of the subunits of the Fcdomain, and the first Fab molecule is fused at the C-terminus of the Fabheavy chain to the N-terminus of the Fab heavy chain of the second Fabmolecule.

In this embodiment, the T cell activating bispecific antigen bindingmolecule essentially comprises an immunoglobulin molecule, wherein inone of the Fab arms the heavy and light chain variable regions VH and VLare exchanged/replaced by each other, and wherein an additional(conventional) Fab molecule is N-terminally fused to said Fab arm (seeFIGS. 1B and 1E). In another such embodiment, the first and the thirdFab molecule are each fused at the C-terminus of the Fab heavy chain tothe N-terminus of one of the subunits of the Fc domain, and the secondFab molecule is fused at the C-terminus of the Fab heavy chain to theN-terminus of the Fab heavy chain of the first Fab molecule. In thisembodiment, the T cell activating bispecific antigen binding moleculeessentially comprises an immunoglobulin molecule with an additional Fabmolecule N-terminally fused to one of the immunoglobulin Fab arms,wherein in said additional Fab molecule the heavy and light chainvariable regions VH and VL are exchanged/replaced by each other (seeFIGS. 1C and 1F).

In all of the different configurations of the T cell activatingbispecific antigen binding molecule according to the invention, theamino acid substitutions described herein may either be in the CH1 andCL domains of the first and (if present) the third Fab molecule, or inthe CH1 and CL domains of the second Fab molecule. Preferably, they arein the CH1 and CL domains of the first and (if present) the third Fabmolecule. In accordance with the concept of the invention, if amino acidsubstitutions as described herein are made in the first (and, ifpresent, the third) Fab molecule, no such amino acid substitutions aremade in the second Fab molecule. Conversely, if amino acid substitutionsas described herein are made in the second Fab molecule, no such aminoacid substitutions are made in the first (and, if present, the third)Fab molecule.

In particular embodiments of the T cell activating bispecific antigenbinding molecule according to the invention, particularly wherein aminoacid substitutions as described herein are made in the first (and, ifpresent, the third) Fab molecule, the constant domain CL of the first(and, if present, the third) Fab molecule is of kappa isotype. In otherembodiments of the T cell activating bispecific antigen binding moleculeaccording to the invention, particularly wherein amino acidsubstitutions as described herein are made in the second Fab molecule,the constant domain CL of the second Fab molecule is of kappa isotype.In some embodiments, the constant domain CL of the first (and, ifpresent, the third) Fab molecule and the constant domain CL of thesecond Fab molecule are of kappa isotype.

In a particular embodiment, the immunoglobulin molecule comprised in theT cell activating bispecific antigen binding molecule according to theinvention is an IgG class immunoglobulin. In an even more particularembodiment the immunoglobulin is an IgG₁ subclass immunoglobulin. Inanother embodiment, the immunoglobulin is an IgG₄ subclassimmunoglobulin.

In a particular embodiment, the invention provides a T cell activatingbispecific antigen binding molecule comprising

-   -   a) a first Fab molecule which specifically binds to a first        antigen;    -   b) a second Fab molecule which specifically binds to a second        antigen, and wherein the variable domains VL and VH of the Fab        light chain and the Fab heavy chain are replaced by each other;    -   c) a third Fab molecule which specifically binds to the first        antigen; and    -   d) an Fc domain composed of a first and a second subunit capable        of stable association;

wherein the first antigen is a target cell antigen and the secondantigen is an activating T cell antigen, particularly CD3, moreparticularly CD3 epsilon;

wherein the third Fab molecule under c) is identical to the first Fabmolecule under a);

wherein in the constant domain CL of the first Fab molecule under a) andthe third Fab molecule under

-   -   c) the amino acid at position 124 is substituted by lysine (K)        (numbering according to Kabat) and the amino acid at position        123 is substituted by lysine (K) or arginine (R) (numbering        according to Kabat), and wherein in the constant domain CH1 of        the first Fab molecule under a) and the third Fab molecule        under c) the amino acid at position 147 is substituted by        glutamic acid (E) (numbering according to Kabat EU index) and        the amino acid at position 213 is substituted by glutamic        acid (E) (numbering according to Kabat EU index); and

wherein

-   -   (i) the first Fab molecule under a) is fused at the C-terminus        of the Fab heavy chain to the N-terminus of the Fab heavy chain        of the second Fab molecule under b), and the second Fab molecule        under b) and the third Fab molecule under c) are each fused at        the C-terminus of the Fab heavy chain to the N-terminus of one        of the subunits of the Fc domain under d), or    -   (ii) the second Fab molecule under b) is fused at the C-terminus        of the Fab heavy chain to the N-terminus of the Fab heavy chain        of the first Fab molecule under a), and the first Fab molecule        under a) and the third Fab molecule under c) are each fused at        the C-terminus of the Fab heavy chain to the N-terminus of one        of the subunits of the Fc domain under d).

In an even more particular embodiment, the invention provides a T cellactivating bispecific antigen binding molecule comprising

-   -   a) a first Fab molecule which specifically binds to a first        antigen;    -   b) a second Fab molecule which specifically binds to a second        antigen, and wherein the variable domains VL and VH of the Fab        light chain and the Fab heavy chain are replaced by each other;    -   c) a third Fab molecule which specifically binds to the first        antigen; and    -   d) an Fc domain composed of a first and a second subunit capable        of stable association; wherein the first antigen is a target        cell antigen and the second antigen is an activating T cell        antigen, particularly CD3, more particularly CD3 epsilon;

wherein the third Fab molecule under c) is identical to the first Fabmolecule under a);

wherein in the constant domain CL of the first Fab molecule under a) andthe third Fab molecule under c) the amino acid at position 124 issubstituted by lysine (K) (numbering according to Kabat) and the aminoacid at position 123 is substituted by arginine (R) (numbering accordingto Kabat), and wherein in the constant domain CH1 of the first Fabmolecule under a) and the third Fab molecule under c) the amino acid atposition 147 is substituted by glutamic acid (E) (numbering according toKabat EU index) and the amino acid at position 213 is substituted byglutamic acid (E) (numbering according to Kabat EU index); and

wherein the first Fab molecule under a) is fused at the C-terminus ofthe Fab heavy chain to the N-terminus of the Fab heavy chain of thesecond Fab molecule under b), and the second Fab molecule under b) andthe third Fab molecule under c) are each fused at the C-terminus of theFab heavy chain to the N-terminus of one of the subunits of the Fcdomain under d).

In a further embodiment, the invention provides a T cell activatingbispecific antigen binding molecule comprising

-   -   a) a first Fab molecule which specifically binds to a first        antigen;    -   b) a second Fab molecule which specifically binds to a second        antigen, and wherein the variable domains VL and VH of the Fab        light chain and the Fab heavy chain are replaced by each other;        and    -   c) an Fc domain composed of a first and a second subunit capable        of stable association;

wherein

-   -   (i) the first antigen is a target cell antigen and the second        antigen is an activating T cell antigen, particularly CD3, more        particularly CD3 epsilon; or    -   (ii) the second antigen is a target cell antigen and the first        antigen is an activating T cell antigen, particularly CD3, more        particularly CD3 epsilon;

wherein in the constant domain CL of the first Fab molecule under a) theamino acid at position 124 is substituted by lysine (K) (numberingaccording to Kabat) and the amino acid at position 123 is substituted bylysine (K) or arginine (R) (numbering according to Kabat), and whereinin the constant domain CH1 of the first Fab molecule under a) the aminoacid at position 147 is substituted by glutamic acid (E) (numberingaccording to Kabat EU index) and the amino acid at position 213 issubstituted by glutamic acid (E) (numbering according to Kabat EUindex); and

wherein the first Fab molecule under a) and the second Fab moleculeunder b) are each fused at the C-terminus of the Fab heavy chain to theN-terminus of one of the subunits of the Fc domain under c).

In particular embodiments of the T cell activating bispecific antigenbinding molecule, the Fc domain is an IgG Fc domain. In a specificembodiment, the Fc domain is an IgG₁ Fc domain. In another specificembodiment, the Fc domain is an IgG₄ Fc domain. In an even more specificembodiment, the Fc domain is an IgG₄ Fc domain comprising the amino acidsubstitution S228P (Kabat numbering). In particular embodiments the Fcdomain is a human Fc domain.

In particular embodiments, the Fc domain comprises a modificationpromoting the association of the first and the second Fc domain subunit.In a specific such embodiment, an amino acid residue in the CH3 domainof the first subunit of the Fc domain is replaced with an amino acidresidue having a larger side chain volume, thereby generating aprotuberance within the CH3 domain of the first subunit which ispositionable in a cavity within the CH3 domain of the second subunit,and an amino acid residue in the CH3 domain of the second subunit of theFc domain is replaced with an amino acid residue having a smaller sidechain volume, thereby generating a cavity within the CH3 domain of thesecond subunit within which the protuberance within the CH3 domain ofthe first subunit is positionable.

In a particular embodiment the Fc domain exhibits reduced bindingaffinity to an Fc receptor and/or reduced effector function, as comparedto a native IgG₁ Fc domain. In certain embodiments the Fc domain isengineered to have reduced binding affinity to an Fc receptor and/orreduced effector function, as compared to a non-engineered Fc domain. Inone embodiment, the Fc domain comprises one or more amino acidsubstitution that reduces binding to an Fc receptor and/or effectorfunction. In one embodiment, the one or more amino acid substitution inthe Fc domain that reduces binding to an Fc receptor and/or effectorfunction is at one or more position selected from the group of L234,L235, and P329 (Kabat EU index numbering). In particular embodiments,each subunit of the Fc domain comprises three amino acid substitutionsthat reduce binding to an Fc receptor and/or effector function whereinsaid amino acid substitutions are L234A, L235A and P329G (Kabat EU indexnumbering). In one such embodiment, the Fc domain is an IgG₁ Fc domain,particularly a human IgG₁ Fc domain. In other embodiments, each subunitof the Fc domain comprises two amino acid substitutions that reducebinding to an Fc receptor and/or effector function wherein said aminoacid substitutions are L235E and P329G (Kabat EU index numbering). Inone such embodiment, the Fc domain is an IgG₄ Fc domain, particularly ahuman IgG₄ Fc domain. In one embodiment, the Fc domain of the T cellactivating bispecific antigen binding molecule is an IgG₄ Fc domain andcomprises the amino acid substitutions L235E and S228P (SPLE) (Kabat EUindex numbering).

In one embodiment the Fc receptor is an Fcγ receptor. In one embodimentthe Fc receptor is a human Fc receptor. In one embodiment, the Fcreceptor is an activating Fc receptor. In a specific embodiment, the Fcreceptor is human FcγRIIa, FcγRI, and/or FcγRIIIa. In one embodiment,the effector function is antibody-dependent cell-mediated cytotoxicity(ADCC).

In a specific embodiment of the T cell activating bispecific antigenbinding molecule according to the invention, the Fab molecule whichspecifically binds to an activating T cell antigen, particularly CD3,more particularly CD3 epsilon, comprises the heavy chain complementaritydetermining region (CDR) 1 of SEQ ID NO: 4, the heavy chain CDR 2 of SEQID NO: 5, the heavy chain CDR 3 of SEQ ID NO: 6, the light chain CDR 1of SEQ ID NO: 8, the light chain CDR 2 of SEQ ID NO: 9 and the lightchain CDR 3 of SEQ ID NO: 10. In an even more specific embodiment, theFab molecule which specifically binds to an activating T cell antigen,particularly CD3, more particularly CD3 epsilon, comprises a heavy chainvariable region comprising an amino acid sequence that is at least about95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence ofSEQ ID NO: 3 and a light chain variable region comprising an amino acidsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 7. In one specificembodiment, the second Fab molecule comprised in the T cell activatingbispecific antigen binding molecule according to the inventionspecifically binds to CD3, more particularly CD3 epsilon, and comprisesthe heavy chain complementarity determining region (CDR) 1 of SEQ ID NO:4, the heavy chain CDR 2 of SEQ ID NO: 5, the heavy chain CDR 3 of SEQID NO: 6, the light chain CDR 1 of SEQ ID NO: 8, the light chain CDR 2of SEQ ID NO: 9 and the light chain CDR 3 of SEQ ID NO: 10. In an evenmore specific embodiment, said second Fab molecule comprises a heavychain variable region comprising the amino acid sequence of SEQ ID NO: 3and a light chain variable region comprising the amino acid sequence ofSEQ ID NO: 7.

In a further specific embodiment of the T cell activating bispecificantigen binding molecule according to the invention, the Fab moleculewhich specifically binds to a target cell antigen, particularly CD20,comprises the heavy chain complementarity determining region (CDR) 1 ofSEQ ID NO: 46, the heavy chain CDR 2 of SEQ ID NO: 47, the heavy chainCDR 3 of SEQ ID NO: 48, the light chain CDR 1 of SEQ ID NO: 49, thelight chain CDR 2 of SEQ ID NO: 50 and the light chain CDR 3 of SEQ IDNO: 51. In an even more specific embodiment, the Fab molecule whichspecifically binds to a target cell antigen, particularly CD20,comprises a heavy chain variable region comprising an amino acidsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 30 and a light chainvariable region comprising an amino acid sequence that is at least about95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence ofSEQ ID NO: 31. In one specific embodiment, the first (and, if present,the third) Fab molecule comprised in the T cell activating bispecificantigen binding molecule according to the invention specifically bindsto CD20, and comprises the heavy chain complementarity determiningregion (CDR) 1 of SEQ ID NO: 46, the heavy chain CDR 2 of SEQ ID NO: 47,the heavy chain CDR 3 of SEQ ID NO: 48, the light chain CDR 1 of SEQ IDNO: 49, the light chain CDR 2 of SEQ ID NO: 50 and the light chain CDR 3of SEQ ID NO: 51. In an even more specific embodiment, said first (and,if present, said third) Fab molecule comprises a heavy chain variableregion comprising the amino acid sequence of SEQ ID NO: 30 and a lightchain variable region comprising the amino acid sequence of SEQ ID NO:31.

In a particular aspect, the invention provides a T cell activatingbispecific antigen binding molecule comprising

-   -   a) a first Fab molecule which specifically binds to a first        antigen;    -   b) a second Fab molecule which specifically binds to a second        antigen, and wherein the variable domains VL and VH of the Fab        light chain and the Fab heavy chain are replaced by each other;    -   c) a third Fab molecule which specifically binds to the first        antigen; and    -   d) an Fc domain composed of a first and a second subunit capable        of stable association;

wherein

-   -   (i) the first antigen is CD20 and the second antigen is CD3,        particularly CD3 epsilon;    -   (ii) the first Fab molecule under a) and the third Fab molecule        under c) each comprise the heavy chain complementarity        determining region (CDR) 1 of SEQ ID NO: 46, the heavy chain CDR        2 of SEQ ID NO: 47, the heavy chain CDR 3 of SEQ ID NO: 48, the        light chain CDR 1 of SEQ ID NO: 49, the light chain CDR 2 of SEQ        ID NO: 50 and the light chain CDR 3 of SEQ ID NO: 51, and the        second Fab molecule under b) comprises the heavy chain CDR 1 of        SEQ ID NO: 4, the heavy chain CDR 2 of SEQ ID NO: 5, the heavy        chain CDR 3 of SEQ ID NO: 6, the light chain CDR 1 of SEQ ID NO:        8, the light chain CDR 2 of SEQ ID NO: 9 and the light chain CDR        3 of SEQ ID NO: 10;    -   (iii) in the constant domain CL of the first Fab molecule        under a) and the third Fab molecule under c) the amino acid at        position 124 is substituted by lysine (K) (numbering according        to Kabat) and the amino acid at position 123 is substituted by        lysine (K) or arginine (R), particularly by arginine (R)        (numbering according to Kabat), and wherein in the constant        domain CH1 of the first Fab molecule under a) and the third Fab        molecule under c) the amino acid at position 147 is substituted        by glutamic acid (E) (numbering according to Kabat EU index) and        the amino acid at position 213 is substituted by glutamic        acid (E) (numbering according to Kabat EU index); and    -   (iv) the first Fab molecule under a) is fused at the C-terminus        of the Fab heavy chain to the N-terminus of the Fab heavy chain        of the second Fab molecule under b), and the second Fab molecule        under b) and the third Fab molecule under c) are each fused at        the C-terminus of the Fab heavy chain to the N-terminus of one        of the subunits of the Fc domain under d).

In a further aspect, the invention provides a T cell activatingbispecific antigen binding molecule comprising

-   -   a) a first Fab molecule which specifically binds to a first        antigen;    -   b) a second Fab molecule which specifically binds to a second        antigen, and wherein the variable domains VL and VH of the Fab        light chain and the Fab heavy chain are replaced by each other;    -   c) a third Fab molecule which specifically binds to the first        antigen; and    -   d) an Fc domain composed of a first and a second subunit capable        of stable association;

wherein

-   -   (i) the first antigen is CD20 and the second antigen is CD3,        particularly CD3 epsilon;    -   (ii) the first Fab molecule under a) and the third Fab molecule        under c) each comprise the heavy chain complementarity        determining region (CDR) 1 of SEQ ID NO: 46, the heavy chain CDR        2 of SEQ ID NO: 47, the heavy chain CDR 3 of SEQ ID NO: 48, the        light chain CDR 1 of SEQ ID NO: 49, the light chain CDR 2 of SEQ        ID NO: 50 and the light chain CDR 3 of SEQ ID NO: 51, and the        second Fab molecule under b) comprises the heavy chain CDR 1 of        SEQ ID NO: 4, the heavy chain CDR 2 of SEQ ID NO: 67, the heavy        chain CDR 3 of SEQ ID NO: 6, the light chain CDR 1 of SEQ ID NO:        68, the light chain CDR 2 of SEQ ID NO: 9 and the light chain        CDR 3 of SEQ ID NO: 10;    -   (iii) in the constant domain CL of the first Fab molecule        under a) and the third Fab molecule under c) the amino acid at        position 124 is substituted by lysine (K) (numbering according        to Kabat) and the amino acid at position 123 is substituted by        lysine (K) or arginine (R), particularly by arginine (R)        (numbering according to Kabat), and wherein in the constant        domain CH1 of the first Fab molecule under a) and the third Fab        molecule under c) the amino acid at position 147 is substituted        by glutamic acid (E) (numbering according to Kabat EU index) and        the amino acid at position 213 is substituted by glutamic        acid (E) (numbering according to Kabat EU index); and    -   (iv) the first Fab molecule under a) is fused at the C-terminus        of the Fab heavy chain to the N-terminus of the Fab heavy chain        of the second Fab molecule under b), and the second Fab molecule        under b) and the third Fab molecule under c) are each fused at        the C-terminus of the Fab heavy chain to the N-terminus of one        of the subunits of the Fc domain under d).

According to another aspect of the invention there is provided one ormore isolated polynucleotide(s) encoding a T cell activating bispecificantigen binding molecule of the invention. The invention furtherprovides one or more expression vector(s) comprising the isolatedpolynucleotide(s) of the invention, and a host cell comprising theisolated polynucleotide(s) or the expression vector(s) of the invention.In some embodiments the host cell is a eukaryotic cell, particularly amammalian cell.

In another aspect is provided a method of producing the T cellactivating bispecific antigen binding molecule of the invention,comprising the steps of a) culturing the host cell of the inventionunder conditions suitable for the expression of the T cell activatingbispecific antigen binding molecule and b) recovering the T cellactivating bispecific antigen binding molecule. The invention alsoencompasses a T cell activating bispecific antigen binding moleculeproduced by the method of the invention.

The invention further provides a pharmaceutical composition comprisingthe T cell activating bispecific antigen binding molecule of theinvention and a pharmaceutically acceptable carrier.

Also encompassed by the invention are methods of using the T cellactivating bispecific antigen binding molecule and pharmaceuticalcomposition of the invention. In one aspect the invention provides a Tcell activating bispecific antigen binding molecule or a pharmaceuticalcomposition of the invention for use as a medicament. In one aspect isprovided a T cell activating bispecific antigen binding molecule or apharmaceutical composition according to the invention for use in thetreatment of a disease in an individual in need thereof. In a specificembodiment the disease is cancer.

Also provided is the use of a T cell activating bispecific antigenbinding molecule of the invention for the manufacture of a medicamentfor the treatment of a disease in an individual in need thereof; as wellas a method of treating a disease in an individual, comprisingadministering to said individual a therapeutically effective amount of acomposition comprising the T cell activating bispecific antigen bindingmolecule according to the invention in a pharmaceutically acceptableform. In a specific embodiment the disease is cancer. In any of theabove embodiments the individual preferably is a mammal, particularly ahuman.

The invention also provides a method for inducing lysis of a targetcell, particularly a tumor cell, comprising contacting a target cellwith a T cell activating bispecific antigen binding molecule of theinvention in the presence of a T cell, particularly a cytotoxic T cell.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-FIG. 1Z. Exemplary configurations of the T cell activatingbispecific antigen binding molecules (TCBs) of the invention. (FIG. 1A,FIG. 1D) Illustration of the “1+1 CrossMab” molecule. (FIG. 1B, FIG. 1E)Illustration of the “2+1 IgG Crossfab” molecule with alternative orderof Crossfab and Fab components (“inverted”). (FIG. 1C, FIG. 1F)Illustration of the “2+1 IgG Crossfab” molecule. (FIG. 1G, FIG. 1K)Illustration of the “1+1 IgG Crossfab” molecule with alternative orderof Crossfab and Fab components (“inverted”). (FIG. 1H, FIG. 1L)Illustration of the “1+1 IgG Crossfab” molecule. (FIG. 1I, FIG. 1M)Illustration of the “2+1 IgG Crossfab” molecule with two CrossFabs.(FIG. 1J, FIG. 1N) Illustration of the “2+1 IgG Crossfab” molecule withtwo CrossFabs and alternative order of Crossfab and Fab components(“inverted”). (FIG. 1O, FIG. 1S) Illustration of the “Fab-Crossfab”molecule. (FIG. 1P, FIG. 1T) Illustration of the “Crossfab-Fab”molecule. (FIG. 1Q, FIG. 1U) Illustration of the “(Fab)₂-Crossfab”molecule. (FIG. 1R, FIG. 1V) Illustration of the “Crossfab-(Fab)₂”molecule. (FIG. 1W, FIG. 1Y) Illustration of the “Fab-(Crossfab)₂”molecule. (FIG. 1X, FIG. 1Z) Illustration of the “(Crossfab)₂-Fab”molecule. Black dot: optional modification in the Fc domain promotingheterodimerization. ++, −−: amino acids of opposite charges introducedin the CH and CL domains.

FIG. 2A-FIG. 2K. Illustration of the TCBs prepared in Example 1. (FIG.2A) “2+1 IgG CrossFab, inverted” without charge modifications (CH1/CLexchange in CD3 binder), (FIG. 2B) “2+1 IgG CrossFab, inverted” withcharge modifications (VH/VL exchange in CD3 binder, charge modificationin CD20 binders, EE=147E, 213E; RK=123R, 124K), (FIG. 2C) “2+1 IgGCrossFab” with charge modifications (VH/VL exchange in CD3 binder,charge modification in CD20 binders, EE=147E, 213E; RK=123R, 124K),(FIG. 2D) “2+1 IgG CrossFab, inverted” without charge modifications(VH/VL exchange in CD3 binder), (FIG. 2E) “2+1 IgG CrossFab, inverted”without charge modifications (VH-CH1/VL-CL exchange in CD3 binder),(FIG. 2F) “2+1 IgG CrossFab, inverted” with charge modifications (VH/VLexchange in CD20 binders, charge modification in CD3 binder, EE=147E,213E; KK=123K, 124K), (FIG. 2G) “2+1 IgG CrossFab, inverted” with chargemodifications and DDKK mutation in Fc region (VH/VL exchange in CD3binder, charge modification in CD20 binders, EE=147E, 213E; RK=123R,124K), (FIG. 2H) “1+1 CrossMab” with charge modifications (VH/VLexchange in CD3 binder, charge modification in CD20 binder, EE=147E,213E; RK=123R, 124K), (FIG. 2I) “1+1 CrossMab” with charge modifications(VH/VL exchange in CD3 binder, charge modification in CD20 binder,EE=147E, 213E; RK=123R, 124K, different CD20 binder), (FIG. 2J) “2+1 IgGCrossFab, inverted” with charge modifications 213E, 123R (VH/VL exchangein CD3 binder, charge modification in CD20 binder, E=213E; R=123R),(FIG. 2K) “2+1 IgG CrossFab, inverted” with charge modifications (VH/VLexchange and charge modification in CD3 binder).

FIG. 3A-FIG. 3Q. (FIG. 3A-FIG. 31 , FIG. 3N, FIG. 3O) CE-SDS analysis ofthe TCBs prepared in Example 1 (final purified preparations). (FIG. 3A)Electropherogram of molecule “A”, shown in FIG. 2A, (FIG. 3B)electropherogram of molecule “B”, shown in FIG. 2B, (FIG. 3C)electropherogram of molecule “C”, shown in FIG. 2C, (FIG. 3D)electropherogram of molecule “D”, shown in FIG. 2D, (FIG. 3E)electropherogram of molecule “E”, shown in FIG. 2E, (FIG. 3F)electropherogram of molecule “F”, shown in FIG. 2F, (FIG. 3G)electropherogram of molecule “G”, shown in FIG. 2G, (FIG. 3H)electropherogram of molecule “H”, shown in FIG. 2H, (FIG. 31 )electropherogram of molecule “I”, shown in FIG. 2I, (FIG. 3N)Electropherogram of molecule “J”, shown in FIG. 2J, (FIG. 3O)electropherogram of molecule “K”, shown in FIG. 2K. Lane A=non-reduced,lane B=reduced. (FIG. 3J-FIG. 3L, FIG. 3P, FIG. 3Q) SDS-PAGE analysis ofTCBs prepared in Example 1 after the first purification step (Protein Aaffinity chromatography). (FIG. 3J) 4-12% Bis-Tris SDS-PAGE, nonreduced; lane 1=marker (Mark 12, unstained standard, Invitrogen); lane2-11=fractions from Protein A affinity chromatography of molecule B,(FIG. 3K) 3-8% Tris-Acetate SDS-PAGE, non reduced; lane 1=marker(HiMark, Invitrogen); lane 2-12=fractions from Protein A affinitychromatography of molecule C, (FIG. 3L) 4-12% Bis-Tris SDS-PAGE, nonreduced; lane 1=marker (Mark 12, unstained standard, Invitrogen); lane2-14=fractions from Protein A affinity chromatography of molecule D,(FIG. 3P) 4-12% Bis/Tris SDS PAGE, non reduced; lane 1=marker (Mark 12,Invitrogen); lane 2-10=fractions from Protein A affinity chromatographyof molecule J, (FIG. 3Q) 4-12% Bis/Tris SDS PAGE, non reduced; lane1=marker (Mark 12, Invitrogen); lane 2-12=fractions from Protein Aaffinity chromatography of molecule K. (FIG. 3M) Preparative sizeexclusion chromatography (SEC; first purification step) of TCBs preparedin Example 1 (molecule A (first SEC step), B and D, as indicated).

FIG. 4 . CD3 and CD20 binding of anti-CD3/anti-CD20 T cell bispecific(TCB) antibodies (“CD20 TCB”) with or without charge modifications(“charge residues”) (see Example 1).

FIG. 5 . Tumor cell lysis induced by anti-CD3/anti-CD20 T cellbispecific (TCB) antibodies (“CD20 TCB”) with or without chargemodifications (“charge residues”) upon 22 h incubation with human PBMCs(see Example 1).

FIG. 6A and FIG. 6B. Activation of CD8⁺ T cells (FIG. 6A) or CD4⁺ Tcells (FIG. 6B) upon T cell-mediated killing of CD20-expressing tumortarget cells (Nalm-6) induced by anti-CD3/anti-CD20 T cell bispecific(TCB) antibodies (“CD20 TCB”) with or without charge modifications(“charge residues”) (see Example 1).

FIG. 7A and FIG. 7B. Activation of CD8⁺ T cells (FIG. 7A) or CD4⁺ Tcells (FIG. 7B) upon T cell-mediated killing of CD20-expressing tumortarget cells (Z-138) induced by anti-CD3/anti-CD20 T cell bispecific(TCB) antibodies (“CD20 TCB”) with or without charge modifications(“charge residues”) (see Example 1).

FIG. 8 . B cell depletion in healthy human whole blood upon incubationwith anti-CD3/anti-CD20 T cell bispecific (TCB) antibodies (“CD20 TCB”)with or without charge modifications (“charge residues”); 22 h assay(see Example 1).

FIG. 9A and FIG. 9B. Activation of CD8⁺ T cells (FIG. 9A) or CD4⁺ Tcells (FIG. 9B) upon T cell-mediated killing of CD20-expressing B cellsin human healthy whole blood induced by anti-CD3/anti-CD20 T cellbispecific (TCB) antibodies (“CD20 TCB”) with or without chargemodifications (“charge residues”) (see Example 1).

FIG. 10A and FIG. 10B. Binding of anti-CD20/anti-CD3 TCB (molecule “B”shown in FIG. 2B) to human CD20- (FIG. 10A) and CD3-expressing (FIG.10B) target cells.

FIG. 11A-FIG. 11C. Binding of anti-CD20/anti-CD3 TCB (molecule “B” shownin FIG. 2B) to human and cynomolgus monkey CD20- and CD3-expressingtarget cells. (FIG. 11A) B-cells, (FIG. 11B) CD4 T cells, (FIG. 11C) CD8T cells.

FIG. 12A and FIG. 12B. Tumor cell lysis mediated by differentanti-CD20/anti-CD3 TCB antibody formats.

FIG. 13A-FIG. 13D. Tumor cell lysis and subsequent T cell activationmediated by different anti-CD20/anti-CD3 TCB antibody formats. (FIG.13A-FIG. 13C) Lysis of Z138 tumor target cells by PBMC effector cellsfrom three different human donors. (FIG. 13D) Lysis of a panel of DLBCLtumor cell lines as indicated.

FIG. 14 . B cell depletion in human whole blood mediated by differentanti-CD20/anti-CD3 TCB antibody formats.

FIG. 15 . Activation of T cells by different anti-CD20/anti-CD3 TCBantibody formats, assessed by quantification of the intensity of CD3downstream signaling using Jurkat-NFAT reporter assay.

FIG. 16 . Pharmacokinetic parameters of a 0.5 mg/kg i.v. bolusadministration of anti-CD20/anti-CD3 TCB antibody (molecule “B” shown inFIG. 2B) from sparse sampling data in NOG mice.

FIG. 17 . Schematic representation of the study design to assess B celldepletion activity of anti-CD20/anti-CD3 TCB antibody (molecule “B”shown in FIG. 2B) in fully humanized NOG mice.

FIG. 18A and FIG. 18B. Kinetics of B-cell and T-cell frequency in bloodof fully humanized NOG mice treated with (FIG. 18B) anti-CD20/anti-CD3TCB antibody (molecule “B” shown in FIG. 2B) or (FIG. 18A) vehiclecontrol. DO, D7: days of therapy injection.

FIG. 19 . Analysis of different surface markers expression on peripheralT-cells three days (D3) and ten days (D10) after vehicle (black bars) oranti-CD20/anti-CD3 TCB antibody (molecule “B” shown in FIG. 2B) (whitebars) injection in fully humanized mice.

FIG. 20A-FIG. 20C. Analysis of B-cell frequency (FIG. 20A), T-cellfrequency (FIG. 20B) and surface markers expression on T-cells (FIG.20C) in spleen of vehicle (black bars) or anti-CD20/anti-CD3 TCBantibody (molecule “B” shown in FIG. 2B) (white bars)-treated fullyhumanized mice at study termination (D10 after first therapeuticinjection).

FIG. 21 . Anti-tumor activity of anti-CD20/anti-CD3 TCB antibody(molecule “B” shown in FIG. 2B) (0.5 mg/kg, once a week) in theWSU-DLCL2 model in NOG mice with huPBMC transfer.

FIG. 22 . Illustration of the “2+1 IgG CrossFab, inverted” moleculesprepared in Example 2. (1) Molecule without charge modifications, (2)molecule with charge modifications in the CH1 and CL domains of the Fabmolecules which specifically bind to BCMA (EE=147E, 213E; KK=123K,124K).

FIG. 23A-FIG. 23C. CE-SDS analysis (lane A=non-reduced, lane B=reduced,peak table for lane A) of “2+1 IgG CrossFab, inverted” molecules used inExample 2. Different methods of purification (Protein A affinitychromatography (PA), size exclusion chromatography (SEC), cationexchange chromatography (cIEX), and a final size exclusionchromatographic step (re-SEC)) were applied for the molecule withoutcharge modifications (83A10-TCB; FIG. 23A and FIG. 23B) and the moleculewith charge modifications (83A10-TCBcv; FIG. 23C).

FIG. 24A-FIG. 24F. CE-SDS analysis (lane A=non-reduced, lane B=reduced,peak table for lane A) of “2+1 IgG CrossFab, inverted” molecules used inExample 2, in head-to-head (H2H) comparison after Protein A affinitychromatography (PA) and size exclusion chromatographic (SEC)purification steps.

FIG. 25A-FIG. 25D. Flow cytometry analysis of binding ofanti-BCMA/anti-CD3 T-cell bispecific antibodies to BCMA-positivemultiple myeloma cell lines. (FIG. 25A) 83A10-TCB on H929 cells andMKN45 cells, (FIG. 25B) 83A10-TCBcv on H929 cells and MKN45 cells, (FIG.25A and FIG. 25D) comparison of 83A10-TCB and 83A10-TCBcv on H929 cells.

FIG. 26A-FIG. 26D. Killing of BCMA-positive H929 myeloma cells byanti-BCMA/anti-CD3 TCB antibodies ((FIG. 26A and FIG. 26B) 83A10-TCB,(FIG. 26C and FIG. 26D) 83A10-TCBcv) as measured by LDH release.

FIG. 27A and FIG. 27B. Illustration of the TCBs prepared in Example 3.(FIG. 27A) “2+1 IgG CrossFab, inverted” with charge modifications (VH/VLexchange in CD3 binder, charge modification in Her2 binders, EE=147E,213E; RK=123R, 124K), (FIG. 27B) “2+1 IgG CrossFab” with chargemodifications (VH/VL exchange in CD3 binder, charge modification in Her3binders, EE=147E, 213E; RK=123R, 124K).

FIG. 28A and FIG. 28B. CE-SDS analysis of the TCBs prepared in Example 3(final purified preparation). (FIG. 28A) Electropherogram of Her2 TCB,shown in FIG. 27A, (FIG. 28B) electropherogram of Her3 TCB, shown inFIG. 27B. Lane A=non-reduced, lane B=reduced.

FIG. 29A and FIG. 29B. Binding of Her2 TCB (FIG. 29A) and Her3 TCB (FIG.29B) to cells, as determined by FACS. Median fluorescence intensitiesfor binding of the Her2 TCB molecule to human CD3 on Jurkat cells (left)or to human Her2 (FIG. 29A) or Her3 (FIG. 29B) on KPL-4 cells (right),as measured by flow cytometry. Depicted are median fluorescence values,based on triplicates, including SD.

FIG. 30 . T cell activation by Her3 TCB. Upon co-incubation of humanPBMC effector cells, KPL-4 target cells and increasing concentrations ofthe Her3 TCB, the percentage of CD69 positive CD8 T cells was measuredby FACS after 48h. Shown are triplicates with SD.

FIG. 31A and FIG. 31B. Activation of Jurkat cells via CD3 after 5h, asdetermined by luminescence. Upon incubation of KPL4 tumor cells withJurkat-NFAT reporter cells (E:T 5:1 (FIG. 31A) or 2.5:1 (FIG. 31B)) andincreasing concentrations of the Her2 TCB (FIG. 31A) or the Her3 TCB(FIG. 31B), activation of Jurkats was determined by relative luminescentsignals (RLUS) after 5h. EC50 values were calculated by Graph Pad Prism(34.4 pM (FIG. 31A) and 22 pM (FIG. 31B)). Depicted are average valuesfrom triplicates, error bars indicate SD.

FIG. 32A-FIG. 32C. (FIG. 32A, FIG. 32B) Tumor cell lysis, as measured byLDH release, upon incubation of Her2-positive KPL4, N87, T47D orMDA-MB-231 target cells with human PBMC effector cells (E:T 10:1) andincreasing concentrations of the Her 2 TCB molecule for 25 h (FIG. 32A)or 46 h (FIG. 32B). Depicted are average values from triplicates, errorbars indicate SD. EC50 values were calculated by GraphPadPrism: 7.5 pM(KPL4 cells), 25.6 pM (N87 cells), 30.6 pM (T47D cells), and 59.9 pM(MDA-MB-231 cells). (FIG. 32C) Tumor cell lysis, as meassured by LDHrelease, upon incubation of Her3-positive KPL4 target cells with humanPBMC effector cells (E:T 10:1) and increasing concentrations of the Her3 TCB molecule for 24 h or 48 h, as indicated. Depicted are averagevalues from triplicates, error bars indicate SD. EC50 values werecalculated by GraphPadPrism: 2.54 pM (24 h) and 0.53 pM (48 h).

FIG. 33 . Tumor cell lysis, induced by Her3 TCB, as determined byCaspase 3/7 activity (luminescence). Shown is the relative luminescentsignal, that was measured as a consequence of Caspase 3/7 activity inKPL-4-Caspase-3/7 GloSensor target cells after 6.5 h co-incubation withPBMCs (E:T=10:1) and different concentrations of Her3 TCB, as indicated.Shown are triplicates with SD. EC50 value was calculated byGraphPadPrism: 0.7 pM.

FIG. 34A and FIG. 34B. Illustration of the TCBs prepared in Example 4.(FIG. 34A) “(Fab)₂-CrossFab” with charge modifications (VH/VL exchangein CD3 binder, charge modification in MCSP binders, EE=147E, 213E;RK=123R, 124K), (FIG. 34B) “(Fab)₂-CrossFab” without chargemodifications (VH/VL exchange in CD3 binder).

FIG. 35 . CE-SDS analysis of the TCB with charge modifications preparedin Example 4 (final purified preparation): Electropherogram of(Fab)₂-XFab-LC007cv, shown in FIG. 34A. Lane A=non-reduced, laneB=reduced.

FIG. 36 . Median fluorescence intensities for binding of the TCBmolecules to human MCSP on MV-3 cells (left) or to human CD3 on Jurkatcells (right), as measured by flow cytometry. Depicted are medianfluorescence values, based on triplicates, including SD.

FIG. 37 . Tumor cell lysis, as measured by LDH release, upon incubationof human MCSP-positive MV-3 cells with human PBMC effector cells (E:T10:1) and increasing concentrations of the TCB molecules for 24h (left)or 48h (right). Depicted are average values from triplicates, error barsindicate SD.

DETAILED DESCRIPTION OF THE INVENTION Definitions

Terms are used herein as generally used in the art, unless otherwisedefined in the following.

As used herein, the term “antigen binding molecule” refers in itsbroadest sense to a molecule that specifically binds an antigenicdeterminant. Examples of antigen binding molecules are immunoglobulinsand derivatives, e.g. fragments, thereof.

The term “bispecific” means that the antigen binding molecule is able tospecifically bind to at least two distinct antigenic determinants.Typically, a bispecific antigen binding molecule comprises two antigenbinding sites, each of which is specific for a different antigenicdeterminant. In certain embodiments the bispecific antigen bindingmolecule is capable of simultaneously binding two antigenicdeterminants, particularly two antigenic determinants expressed on twodistinct cells.

The term “valent” as used herein denotes the presence of a specifiednumber of antigen binding sites in an antigen binding molecule. As such,the term “monovalent binding to an antigen” denotes the presence of one(and not more than one) antigen binding site specific for the antigen inthe antigen binding molecule.

An “antigen binding site” refers to the site, i.e. one or more aminoacid residues, of an antigen binding molecule which provides interactionwith the antigen. For example, the antigen binding site of an antibodycomprises amino acid residues from the complementarity determiningregions (CDRs). A native immunoglobulin molecule typically has twoantigen binding sites, a Fab molecule typically has a single antigenbinding site.

As used herein, the term “antigen binding moiety” refers to apolypeptide molecule that specifically binds to an antigenicdeterminant. In one embodiment, an antigen binding moiety is able todirect the entity to which it is attached (e.g. a second antigen bindingmoiety) to a target site, for example to a specific type of tumor cellor tumor stroma bearing the antigenic determinant. In another embodimentan antigen binding moiety is able to activate signaling through itstarget antigen, for example a T cell receptor complex antigen. Antigenbinding moieties include antibodies and fragments thereof as furtherdefined herein. Particular antigen binding moieties include an antigenbinding domain of an antibody, comprising an antibody heavy chainvariable region and an antibody light chain variable region. In certainembodiments, the antigen binding moieties may comprise antibody constantregions as further defined herein and known in the art. Useful heavychain constant regions include any of the five isotypes: α, δ, ε, γ, orμ. Useful light chain constant regions include any of the two isotypes:κ and λ.

As used herein, the term “antigenic determinant” is synonymous with“antigen” and “epitope,” and refers to a site (e.g. a contiguous stretchof amino acids or a conformational configuration made up of differentregions of non-contiguous amino acids) on a polypeptide macromolecule towhich an antigen binding moiety binds, forming an antigen bindingmoiety-antigen complex. Useful antigenic determinants can be found, forexample, on the surfaces of tumor cells, on the surfaces ofvirus-infected cells, on the surfaces of other diseased cells, on thesurface of immune cells, free in blood serum, and/or in theextracellular matrix (ECM). The proteins referred to as antigens herein(e.g. CD3) can be any native form the proteins from any vertebratesource, including mammals such as primates (e.g. humans) and rodents(e.g. mice and rats), unless otherwise indicated. In a particularembodiment the antigen is a human protein. Where reference is made to aspecific protein herein, the term encompasses the “full-length”,unprocessed protein as well as any form of the protein that results fromprocessing in the cell. The term also encompasses naturally occurringvariants of the protein, e.g. splice variants or allelic variants. Anexemplary human protein useful as antigen is CD3, particularly theepsilon subunit of CD3 (see UniProt no. P07766 (version 130), NCBIRefSeq no. NP_000724.1, SEQ ID NO: 1 for the human sequence; or UniProtno. Q95LI5 (version 49), NCBI GenBank no. BAB71849.1, SEQ ID NO: 2 forthe cynomolgus [Macaca fascicularis] sequence). In certain embodimentsthe T cell activating bispecific antigen binding molecule of theinvention binds to an epitope of CD3 or a target cell antigen that isconserved among the CD3 or target cell antigen from different species.

By “specific binding” is meant that the binding is selective for theantigen and can be discriminated from unwanted or non-specificinteractions. The ability of an antigen binding moiety to bind to aspecific antigenic determinant can be measured either through anenzyme-linked immunosorbent assay (ELISA) or other techniques familiarto one of skill in the art, e.g. surface plasmon resonance (SPR)technique (analyzed on a BIAcore instrument) (Liljeblad et al., Glyco J17, 323-329 (2000)), and traditional binding assays (Heeley, Endocr Res28, 217-229 (2002)). In one embodiment, the extent of binding of anantigen binding moiety to an unrelated protein is less than about 10% ofthe binding of the antigen binding moiety to the antigen as measured,e.g., by SPR. In certain embodiments, an antigen binding moiety thatbinds to the antigen, or an antigen binding molecule comprising thatantigen binding moiety, has a dissociation constant (K_(D)) of ≤1 μM,≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g. 10⁻⁸M orless, e.g. from 10⁻⁸M to 10⁻¹³ M, e.g., from 10⁻⁹M to 10⁻¹³ M).“Affinity” refers to the strength of the sum total of non-covalentinteractions between a single binding site of a molecule (e.g., areceptor) and its binding partner (e.g., a ligand). Unless indicatedotherwise, as used herein, “binding affinity” refers to intrinsicbinding affinity which reflects a 1:1 interaction between members of abinding pair (e.g., an antigen binding moiety and an antigen, or areceptor and its ligand). The affinity of a molecule X for its partner Ycan generally be represented by the dissociation constant (K_(D)), whichis the ratio of dissociation and association rate constants (k_(off) andk_(on), respectively). Thus, equivalent affinities may comprisedifferent rate constants, as long as the ratio of the rate constantsremains the same. Affinity can be measured by well established methodsknown in the art, including those described herein. A particular methodfor measuring affinity is Surface Plasmon Resonance (SPR).

“Reduced binding”, for example reduced binding to an Fc receptor, refersto a decrease in affinity for the respective interaction, as measuredfor example by SPR. For clarity the term includes also reduction of theaffinity to zero (or below the detection limit of the analytic method),i.e. complete abolishment of the interaction. Conversely, “increasedbinding” refers to an increase in binding affinity for the respectiveinteraction.

An “activating T cell antigen” as used herein refers to an antigenicdeterminant expressed on the surface of a T lymphocyte, particularly acytotoxic T lymphocyte, which is capable of inducing T cell activationupon interaction with an antigen binding molecule. Specifically,interaction of an antigen binding molecule with an activating T cellantigen may induce T cell activation by triggering the signaling cascadeof the T cell receptor complex. In a particular embodiment theactivating T cell antigen is CD3, particularly the epsilon subunit ofCD3 (see UniProt no. P07766 (version 130), NCBI RefSeq no. NP_000724.1,SEQ ID NO: 1 for the human sequence; or UniProt no. Q95LI5 (version 49),NCBI GenBank no. BAB71849.1, SEQ ID NO: 2 for the cynomolgus [Macacafascicularis] sequence).

“T cell activation” as used herein refers to one or more cellularresponse of a T lymphocyte, particularly a cytotoxic T lymphocyte,selected from: proliferation, differentiation, cytokine secretion,cytotoxic effector molecule release, cytotoxic activity, and expressionof activation markers. The T cell activating bispecific antigen bindingmolecules of the invention are capable of inducing T cell activation.Suitable assays to measure T cell activation are known in the artdescribed herein.

A “target cell antigen” as used herein refers to an antigenicdeterminant presented on the surface of a target cell, for example acell in a tumor such as a cancer cell or a cell of the tumor stroma. Ina particular embodiment, the target cell antigen is CD20, particularlyhuman CD20 (see UniProt no. P11836).

As used herein, the terms “first”, “second” or “third” with respect toFab molecules etc., are used for convenience of distinguishing whenthere is more than one of each type of moiety. Use of these terms is notintended to confer a specific order or orientation of the T cellactivating bispecific antigen binding molecule unless explicitly sostated.

A “Fab molecule” refers to a protein consisting of the VH and CH1 domainof the heavy chain (the “Fab heavy chain”) and the VL and CL domain ofthe light chain (the “Fab light chain”) of an immunoglobulin.

By “fused” is meant that the components (e.g. a Fab molecule and an Fcdomain subunit) are linked by peptide bonds, either directly or via oneor more peptide linkers.

As used herein, the term “single-chain” refers to a molecule comprisingamino acid monomers linearly linked by peptide bonds. In certainembodiments, one of the antigen binding moieties is a single-chain Fabmolecule, i.e. a Fab molecule wherein the Fab light chain and the Fabheavy chain are connected by a peptide linker to form a single peptidechain. In a particular such embodiment, the C-terminus of the Fab lightchain is connected to the N-terminus of the Fab heavy chain in thesingle-chain Fab molecule.

By a “crossover” Fab molecule (also termed “Crossfab”) is meant a Fabmolecule wherein the variable domains of the Fab heavy and light chainare exchanged (i.e. replaced by each other), i.e. the crossover Fabmolecule comprises a peptide chain composed of the light chain variabledomain VL and the heavy chain constant domain 1 CH1 (VL-CH1, in N- toC-terminal direction), and a peptide chain composed of the heavy chainvariable domain VH and the light chain constant domain CL (VH-CL, in N-to C-terminal direction). For clarity, in a crossover Fab moleculewherein the variable domains of the Fab light chain and the Fab heavychain are exchanged, the peptide chain comprising the heavy chainconstant domain 1 CH1 is referred to herein as the “heavy chain” of thecrossover Fab molecule. In contrast thereto, by a “conventional” Fabmolecule is meant a Fab molecule in its natural format, i.e. comprisinga heavy chain composed of the heavy chain variable and constant domains(VH-CH1, in N- to C-terminal direction), and a light chain composed ofthe light chain variable and constant domains (VL-CL, in N- toC-terminal direction).

The term “immunoglobulin molecule” refers to a protein having thestructure of a naturally occurring antibody. For example,immunoglobulins of the IgG class are heterotetrameric glycoproteins ofabout 150,000 daltons, composed of two light chains and two heavy chainsthat are disulfide-bonded. From N- to C-terminus, each heavy chain has avariable domain (VH), also called a variable heavy domain or a heavychain variable region, followed by three constant domains (CH1, CH2, andCH3), also called a heavy chain constant region. Similarly, from N- toC-terminus, each light chain has a variable domain (VL), also called avariable light domain or a light chain variable region, followed by aconstant light (CL) domain, also called a light chain constant region.The heavy chain of an immunoglobulin may be assigned to one of fivetypes, called α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), some ofwhich may be further divided into subtypes, e.g. γ₁ (IgG₁), γ₂ (IgG₂),γ₃ (IgG₃), γ₄ (IgG₄), α₁ (IgA₁) and α₂ (IgA₂). The light chain of animmunoglobulin may be assigned to one of two types, called kappa (κ) andlambda (λ), based on the amino acid sequence of its constant domain. Animmunoglobulin essentially consists of two Fab molecules and an Fcdomain, linked via the immunoglobulin hinge region.

The term “antibody” herein is used in the broadest sense and encompassesvarious antibody structures, including but not limited to monoclonalantibodies, polyclonal antibodies, and antibody fragments so long asthey exhibit the desired antigen-binding activity.

An “antibody fragment” refers to a molecule other than an intactantibody that comprises a portion of an intact antibody that binds theantigen to which the intact antibody binds. Examples of antibodyfragments include but are not limited to Fv, Fab, Fab′, Fab′-SH,F(ab′)₂, diabodies, linear antibodies, single-chain antibody molecules(e.g. scFv), and single-domain antibodies. For a review of certainantibody fragments, see Hudson et al., Nat Med 9, 129-134 (2003). For areview of scFv fragments, see e.g. Plückthun, in The Pharmacology ofMonoclonal Antibodies, vol. 113, Rosenburg and Moore eds.,Springer-Verlag, New York, pp. 269-315 (1994); see also WO 93/16185; andU.S. Pat. Nos. 5,571,894 and 5,587,458. For discussion of Fab andF(ab′)₂ fragments comprising salvage receptor binding epitope residuesand having increased in vivo half-life, see U.S. Pat. No. 5,869,046.Diabodies are antibody fragments with two antigen-binding sites that maybe bivalent or bispecific. See, for example, EP 404,097; WO 1993/01161;Hudson et al., Nat Med 9, 129-134 (2003); and Hollinger et al., ProcNatl Acad Sci USA 90, 6444-6448 (1993). Triabodies and tetrabodies arealso described in Hudson et al., Nat Med 9, 129-134 (2003).Single-domain antibodies are antibody fragments comprising all or aportion of the heavy chain variable domain or all or a portion of thelight chain variable domain of an antibody. In certain embodiments, asingle-domain antibody is a human single-domain antibody (Domantis,Inc., Waltham, Mass.; see e.g. U.S. Pat. No. 6,248,516 B1). Antibodyfragments can be made by various techniques, including but not limitedto proteolytic digestion of an intact antibody as well as production byrecombinant host cells (e.g. E. coli or phage), as described herein.

The term “antigen binding domain” refers to the part of an antibody thatcomprises the area which specifically binds to and is complementary topart or all of an antigen. An antigen binding domain may be provided by,for example, one or more antibody variable domains (also called antibodyvariable regions). Particularly, an antigen binding domain comprises anantibody light chain variable domain (VL) and an antibody heavy chainvariable domain (VH).

The term “variable region” or “variable domain” refers to the domain ofan antibody heavy or light chain that is involved in binding theantibody to antigen. The variable domains of the heavy chain and lightchain (VH and VL, respectively) of a native antibody generally havesimilar structures, with each domain comprising four conserved frameworkregions (FRs) and three hypervariable regions (HVRs). See, e.g., Kindtet al., Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007).A single VH or VL domain may be sufficient to confer antigen-bindingspecificity.

The term “hypervariable region” or “HVR”, as used herein, refers to eachof the regions of an antibody variable domain which are hypervariable insequence and/or form structurally defined loops (“hypervariable loops”).Generally, native four-chain antibodies comprise six HVRs; three in theVH (H1, H2, H3), and three in the VL (L1, L2, L3). HVRs generallycomprise amino acid residues from the hypervariable loops and/or fromthe complementarity determining regions (CDRs), the latter being ofhighest sequence variability and/or involved in antigen recognition.With the exception of CDR1 in VH, CDRs generally comprise the amino acidresidues that form the hypervariable loops. Hypervariable regions (HVRs)are also referred to as “complementarity determining regions” (CDRs),and these terms are used herein interchangeably in reference to portionsof the variable region that form the antigen binding regions. Thisparticular region has been described by Kabat et al., Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991) and by Chothia etal., J Mol Biol 196:901-917 (1987), where the definitions includeoverlapping or subsets of amino acid residues when compared against eachother. Nevertheless, application of either definition to refer to a CDRof an antibody or variants thereof is intended to be within the scope ofthe term as defined and used herein. The appropriate amino acid residueswhich encompass the CDRs as defined by each of the above citedreferences are set forth below in Table 1 as a comparison. The exactresidue numbers which encompass a particular CDR will vary depending onthe sequence and size of the CDR. Those skilled in the art can routinelydetermine which residues comprise a particular CDR given the variableregion amino acid sequence of the antibody. The CDR sequences givenherein are generally according to the Kabat definition.

TABLE 1 CDR Definitions¹ CDR Kabat Chothia AbM² V_(H) CDR1 31-35 26-3226-35 V_(H) CDR2 50-65 52-58 50-58 V_(H) CDR3  95-102  95-102  95-102V_(L) CDR1 24-34 26-32 24-34 V_(L) CDR2 50-56 50-52 50-56 V_(L) CDR389-97 91-96 89-97 ¹Numbering of all CDR definitions in Table 1 isaccording to the numbering conventions set forth by Kabat et al. (seebelow). ²“AbM” with a lowercase “b” as used in Table 1 refers to theCDRs as defined by Oxford Molecular's “AbM” antibody modeling software.

Kabat et al. also defined a numbering system for variable regionsequences that is applicable to any antibody. One of ordinary skill inthe art can unambiguously assign this system of “Kabat numbering” to anyvariable region sequence, without reliance on any experimental databeyond the sequence itself. As used herein in connection with variableregion seqeunces, “Kabat numbering” refers to the numbering system setforth by Kabat et al., Sequences of Proteins of Immunological Interest,5th Ed. Public Health Service, National Institutes of Health, Bethesda,Md. (1991). Unless otherwise specified, references to the numbering ofspecific amino acid residue positions in an antibody variable region areaccording to the Kabat numbering system.

As used herein, the amino acid positions of all constant regions anddomains of the heavy and light chain are numbered according to the Kabatnumbering system described in Kabat, et al., Sequences of Proteins ofImmunological Interest, 5th ed., Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991) and is referred to as“numbering according to Kabat” or “Kabat numbering” herein. Specificallythe Kabat numbering system (see pages 647-660 of Kabat, et al.,Sequences of Proteins of Immunological Interest, 5th ed., Public HealthService, National Institutes of Health, Bethesda, Md. (1991)) is usedfor the light chain constant domain CL of kappa and lambda isotype andthe Kabat EU index numbering system (see pages 661-723) is used for theheavy chain constant domains (CH1, Hinge, CH2 and CH3), which is hereinfurther clarified by referring to “numbering according to Kabat EUindex” in this case.

The polypeptide sequences of the sequence listing are not numberedaccording to the Kabat numbering system. However, it is well within theordinary skill of one in the art to convert the numbering of thesequences of the Sequence Listing to Kabat numbering.

“Framework” or “FR” refers to variable domain residues other thanhypervariable region (HVR) residues. The FR of a variable domaingenerally consists of four FR domains: FR1, FR2, FR3, and FR4.Accordingly, the HVR and FR sequences generally appear in the followingsequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

The “class” of an antibody or immunoglobulin refers to the type ofconstant domain or constant region possessed by its heavy chain. Thereare five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, andseveral of these may be further divided into subclasses (isotypes),e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁, and IgA₂. The heavy chain constantdomains that correspond to the different classes of immunoglobulins arecalled α, δ, ε, γ, and μ, respectively.

The term “Fc domain” or “Fc region” herein is used to define aC-terminal region of an immunoglobulin heavy chain that contains atleast a portion of the constant region. The term includes nativesequence Fc regions and variant Fc regions. Although the boundaries ofthe Fc region of an IgG heavy chain might vary slightly, the human IgGheavy chain Fc region is usually defined to extend from Cys226, or fromPro230, to the carboxyl-terminus of the heavy chain. However, antibodiesproduced by host cells may undergo post-translational cleavage of one ormore, particularly one or two, amino acids from the C-terminus of theheavy chain. Therefore an antibody produced by a host cell by expressionof a specific nucleic acid molecule encoding a full-length heavy chainmay include the full-length heavy chain, or it may include a cleavedvariant of the full-length heavy chain (also referred to herein as a“cleaved variant heavy chain”). This may be the case where the final twoC-terminal amino acids of the heavy chain are glycine (G446) and lysine(K447, numbering according to Kabat EU index). Therefore, the C-terminallysine (Lys447), or the C-terminal glycine (Gly446) and lysine (K447),of the Fc region may or may not be present. Amino acid sequences ofheavy chains including Fc domains (or a subunit of an Fc domain asdefined herein) are denoted herein without C-terminal glycine-lysinedipeptide if not indicated otherwise. In one embodiment of theinvention, a heavy chain including a subunit of an Fc domain asspecified herein, comprised in a T cell activating bispecific antigenbinding molecule according to the invention, comprises an additionalC-terminal glycine-lysine dipeptide (G446 and K447, numbering accordingto EU index of Kabat). In one embodiment of the invention, a heavy chainincluding a subunit of an Fc domain as specified herein, comprised in aT cell activating bispecific antigen binding molecule according to theinvention, comprises an additional C-terminal glycine residue (G446,numbering according to EU index of Kabat). Compositions of theinvention, such as the pharmaceutical compositions described herein,comprise a population of T cell activating bispecific antigen bindingmolecules of the invention. The population of T cell activatingbispecific antigen binding molecule may comprise molecules having afull-length heavy chain and molecules having a cleaved variant heavychain. The population of T cell activating bispecific antigen bindingmolecules may consist of a mixture of molecules having a full-lengthheavy chain and molecules having a cleaved variant heavy chain, whereinat least 50%, at least 60%, at least 70%, at least 80% or at least 90%of the T cell activating bispecific antigen binding molecules have acleaved variant heavy chain. In one embodiment of the invention acomposition comprising a population of T cell activating bispecificantigen binding molecules of the invention comprises an T cellactivating bispecific antigen binding molecule comprising a heavy chainincluding a subunit of an Fc domain as specified herein with anadditional C-terminal glycine-lysine dipeptide (G446 and K447, numberingaccording to EU index of Kabat). In one embodiment of the invention acomposition comprising a population of T cell activating bispecificantigen binding molecules of the invention comprises an T cellactivating bispecific antigen binding molecule comprising a heavy chainincluding a subunit of an Fc domain as specified herein with anadditional C-terminal glycine residue (G446, numbering according to EUindex of Kabat). In one embodiment of the invention such a compositioncomprises a population of T cell activating bispecific antigen bindingmolecules comprised of molecules comprising a heavy chain including asubunit of an Fc domain as specified herein; molecules comprising aheavy chain including a subunit of a Fc domain as specified herein withan additional C-terminal glycine residue (G446, numbering according toEU index of Kabat); and molecules comprising a heavy chain including asubunit of an Fc domain as specified herein with an additionalC-terminal glycine-lysine dipeptide (G446 and K447, numbering accordingto EU index of Kabat). Unless otherwise specified herein, numbering ofamino acid residues in the Fc region or constant region is according tothe EU numbering system, also called the EU index, as described in Kabatet al., Sequences of Proteins of Immunological Interest, 5th Ed. PublicHealth Service, National Institutes of Health, Bethesda, Md., 1991 (seealso above). A “subunit” of an Fc domain as used herein refers to one ofthe two polypeptides forming the dimeric Fc domain, i.e. a polypeptidecomprising C-terminal constant regions of an immunoglobulin heavy chain,capable of stable self-association. For example, a subunit of an IgG Fcdomain comprises an IgG CH2 and an IgG CH3 constant domain.

A “modification promoting the association of the first and the secondsubunit of the Fc domain” is a manipulation of the peptide backbone orthe post-translational modifications of an Fc domain subunit thatreduces or prevents the association of a polypeptide comprising the Fcdomain subunit with an identical polypeptide to form a homodimer. Amodification promoting association as used herein particularly includesseparate modifications made to each of the two Fc domain subunitsdesired to associate (i.e. the first and the second subunit of the Fcdomain), wherein the modifications are complementary to each other so asto promote association of the two Fc domain subunits. For example, amodification promoting association may alter the structure or charge ofone or both of the Fc domain subunits so as to make their associationsterically or electrostatically favorable, respectively. Thus,(hetero)dimerization occurs between a polypeptide comprising the firstFc domain subunit and a polypeptide comprising the second Fc domainsubunit, which might be non-identical in the sense that furthercomponents fused to each of the subunits (e.g. antigen binding moieties)are not the same. In some embodiments the modification promotingassociation comprises an amino acid mutation in the Fc domain,specifically an amino acid substitution. In a particular embodiment, themodification promoting association comprises a separate amino acidmutation, specifically an amino acid substitution, in each of the twosubunits of the Fc domain.

The term “effector functions” refers to those biological activitiesattributable to the Fc region of an antibody, which vary with theantibody isotype. Examples of antibody effector functions include: C1qbinding and complement dependent cytotoxicity (CDC), Fc receptorbinding, antibody-dependent cell-mediated cytotoxicity (ADCC),antibody-dependent cellular phagocytosis (ADCP), cytokine secretion,immune complex-mediated antigen uptake by antigen presenting cells, downregulation of cell surface receptors (e.g. B cell receptor), and B cellactivation.

As used herein, the terms “engineer, engineered, engineering”, areconsidered to include any manipulation of the peptide backbone or thepost-translational modifications of a naturally occurring or recombinantpolypeptide or fragment thereof. Engineering includes modifications ofthe amino acid sequence, of the glycosylation pattern, or of the sidechain group of individual amino acids, as well as combinations of theseapproaches.

The term “amino acid mutation” as used herein is meant to encompassamino acid substitutions, deletions, insertions, and modifications. Anycombination of substitution, deletion, insertion, and modification canbe made to arrive at the final construct, provided that the finalconstruct possesses the desired characteristics, e.g., reduced bindingto an Fc receptor, or increased association with another peptide. Aminoacid sequence deletions and insertions include amino- and/orcarboxy-terminal deletions and insertions of amino acids. Particularamino acid mutations are amino acid substitutions. For the purpose ofaltering e.g. the binding characteristics of an Fc region,non-conservative amino acid substitutions, i.e. replacing one amino acidwith another amino acid having different structural and/or chemicalproperties, are particularly preferred. Amino acid substitutions includereplacement by non-naturally occurring amino acids or by naturallyoccurring amino acid derivatives of the twenty standard amino acids(e.g. 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine,5-hydroxylysine). Amino acid mutations can be generated using genetic orchemical methods well known in the art. Genetic methods may includesite-directed mutagenesis, PCR, gene synthesis and the like. It iscontemplated that methods of altering the side chain group of an aminoacid by methods other than genetic engineering, such as chemicalmodification, may also be useful. Various designations may be usedherein to indicate the same amino acid mutation. For example, asubstitution from proline at position 329 of the Fc domain to glycinecan be indicated as 329G, G329, G329, P329, or Pro329Gly.

As used herein, term “polypeptide” refers to a molecule composed ofmonomers (amino acids) linearly linked by amide bonds (also known aspeptide bonds). The term “polypeptide” refers to any chain of two ormore amino acids, and does not refer to a specific length of theproduct. Thus, peptides, dipeptides, tripeptides, oligopeptides,“protein,” “amino acid chain,” or any other term used to refer to achain of two or more amino acids, are included within the definition of“polypeptide,” and the term “polypeptide” may be used instead of, orinterchangeably with any of these terms. The term “polypeptide” is alsointended to refer to the products of post-expression modifications ofthe polypeptide, including without limitation glycosylation,acetylation, phosphorylation, amidation, derivatization by knownprotecting/blocking groups, proteolytic cleavage, or modification bynon-naturally occurring amino acids. A polypeptide may be derived from anatural biological source or produced by recombinant technology, but isnot necessarily translated from a designated nucleic acid sequence. Itmay be generated in any manner, including by chemical synthesis. Apolypeptide of the invention may be of a size of about 3 or more, 5 ormore, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 ormore, 200 or more, 500 or more, 1,000 or more, or 2,000 or more aminoacids. Polypeptides may have a defined three-dimensional structure,although they do not necessarily have such structure. Polypeptides witha defined three-dimensional structure are referred to as folded, andpolypeptides which do not possess a defined three-dimensional structure,but rather can adopt a large number of different conformations, and arereferred to as unfolded.

By an “isolated” polypeptide or a variant, or derivative thereof isintended a polypeptide that is not in its natural milieu. No particularlevel of purification is required. For example, an isolated polypeptidecan be removed from its native or natural environment. Recombinantlyproduced polypeptides and proteins expressed in host cells areconsidered isolated for the purpose of the invention, as are native orrecombinant polypeptides which have been separated, fractionated, orpartially or substantially purified by any suitable technique. “Percent(%) amino acid sequence identity” with respect to a referencepolypeptide sequence is defined as the percentage of amino acid residuesin a candidate sequence that are identical with the amino acid residuesin the reference polypeptide sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor aligning sequences, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.For purposes herein, however, % amino acid sequence identity values aregenerated using the sequence comparison computer program ALIGN-2. TheALIGN-2 sequence comparison computer program was authored by Genentech,Inc., and the source code has been filed with user documentation in theU.S. Copyright Office, Washington D.C., 20559, where it is registeredunder U.S. Copyright Registration No. TXU510087. The ALIGN-2 program ispublicly available from Genentech, Inc., South San Francisco, Calif., ormay be compiled from the source code. The ALIGN-2 program should becompiled for use on a UNIX operating system, including digital UNIXV4.0D. All sequence comparison parameters are set by the ALIGN-2 programand do not vary. In situations where ALIGN-2 is employed for amino acidsequence comparisons, the % amino acid sequence identity of a givenamino acid sequence A to, with, or against a given amino acid sequence B(which can alternatively be phrased as a given amino acid sequence Athat has or comprises a certain % amino acid sequence identity to, with,or against a given amino acid sequence B) is calculated as follows:

100 times the fraction X/Y

where X is the number of amino acid residues scored as identical matchesby the sequence alignment program ALIGN-2 in that program's alignment ofA and B, and where Y is the total number of amino acid residues in B. Itwill be appreciated that where the length of amino acid sequence A isnot equal to the length of amino acid sequence B, the % amino acidsequence identity of A to B will not equal the % amino acid sequenceidentity of B to A. Unless specifically stated otherwise, all % aminoacid sequence identity values used herein are obtained as described inthe immediately preceding paragraph using the ALIGN-2 computer program.

The term “polynucleotide” refers to an isolated nucleic acid molecule orconstruct, e.g. messenger RNA (mRNA), virally-derived RNA, or plasmidDNA (pDNA). A polynucleotide may comprise a conventional phosphodiesterbond or a non-conventional bond (e.g. an amide bond, such as found inpeptide nucleic acids (PNA). The term “nucleic acid molecule” refers toany one or more nucleic acid segments, e.g. DNA or RNA fragments,present in a polynucleotide.

By “isolated” nucleic acid molecule or polynucleotide is intended anucleic acid molecule, DNA or RNA, which has been removed from itsnative environment. For example, a recombinant polynucleotide encoding apolypeptide contained in a vector is considered isolated for thepurposes of the present invention. Further examples of an isolatedpolynucleotide include recombinant polynucleotides maintained inheterologous host cells or purified (partially or substantially)polynucleotides in solution. An isolated polynucleotide includes apolynucleotide molecule contained in cells that ordinarily contain thepolynucleotide molecule, but the polynucleotide molecule is presentextrachromosomally or at a chromosomal location that is different fromits natural chromosomal location. Isolated RNA molecules include in vivoor in vitro RNA transcripts of the present invention, as well aspositive and negative strand forms, and double-stranded forms. Isolatedpolynucleotides or nucleic acids according to the present inventionfurther include such molecules produced synthetically. In addition, apolynucleotide or a nucleic acid may be or may include a regulatoryelement such as a promoter, ribosome binding site, or a transcriptionterminator.

By a nucleic acid or polynucleotide having a nucleotide sequence atleast, for example, 95% “identical” to a reference nucleotide sequenceof the present invention, it is intended that the nucleotide sequence ofthe polynucleotide is identical to the reference sequence except thatthe polynucleotide sequence may include up to five point mutations pereach 100 nucleotides of the reference nucleotide sequence. In otherwords, to obtain a polynucleotide having a nucleotide sequence at least95% identical to a reference nucleotide sequence, up to 5% of thenucleotides in the reference sequence may be deleted or substituted withanother nucleotide, or a number of nucleotides up to 5% of the totalnucleotides in the reference sequence may be inserted into the referencesequence. These alterations of the reference sequence may occur at the5′ or 3′ terminal positions of the reference nucleotide sequence oranywhere between those terminal positions, interspersed eitherindividually among residues in the reference sequence or in one or morecontiguous groups within the reference sequence. As a practical matter,whether any particular polynucleotide sequence is at least 80%, 85%,90%, 95%, 96%, 97%, 98% or 99% identical to a nucleotide sequence of thepresent invention can be determined conventionally using known computerprograms, such as the ones discussed above for polypeptides (e.g.ALIGN-2).

The term “expression cassette” refers to a polynucleotide generatedrecombinantly or synthetically, with a series of specified nucleic acidelements that permit transcription of a particular nucleic acid in atarget cell. The recombinant expression cassette can be incorporatedinto a plasmid, chromosome, mitochondrial DNA, plastid DNA, virus, ornucleic acid fragment. Typically, the recombinant expression cassetteportion of an expression vector includes, among other sequences, anucleic acid sequence to be transcribed and a promoter. In certainembodiments, the expression cassette of the invention comprisespolynucleotide sequences that encode bispecific antigen bindingmolecules of the invention or fragments thereof.

The term “vector” or “expression vector” is synonymous with “expressionconstruct” and refers to a DNA molecule that is used to introduce anddirect the expression of a specific gene to which it is operablyassociated in a target cell. The term includes the vector as aself-replicating nucleic acid structure as well as the vectorincorporated into the genome of a host cell into which it has beenintroduced. The expression vector of the present invention comprises anexpression cassette. Expression vectors allow transcription of largeamounts of stable mRNA. Once the expression vector is inside the targetcell, the ribonucleic acid molecule or protein that is encoded by thegene is produced by the cellular transcription and/or translationmachinery. In one embodiment, the expression vector of the inventioncomprises an expression cassette that comprises polynucleotide sequencesthat encode bispecific antigen binding molecules of the invention orfragments thereof.

The terms “host cell”, “host cell line,” and “host cell culture” areused interchangeably and refer to cells into which exogenous nucleicacid has been introduced, including the progeny of such cells. Hostcells include “transformants” and “transformed cells,” which include theprimary transformed cell and progeny derived therefrom without regard tothe number of passages. Progeny may not be completely identical innucleic acid content to a parent cell, but may contain mutations. Mutantprogeny that have the same function or biological activity as screenedor selected for in the originally transformed cell are included herein.A host cell is any type of cellular system that can be used to generatethe bispecific antigen binding molecules of the present invention. Hostcells include cultured cells, e.g. mammalian cultured cells, such as CHOcells, BHK cells, NS0 cells, SP2/0 cells, YO myeloma cells, P3X63 mousemyeloma cells, PER cells, PER.C6 cells or hybridoma cells, yeast cells,insect cells, and plant cells, to name only a few, but also cellscomprised within a transgenic animal, transgenic plant or cultured plantor animal tissue.

An “activating Fc receptor” is an Fc receptor that following engagementby an Fc domain of an antibody elicits signaling events that stimulatethe receptor-bearing cell to perform effector functions. Humanactivating Fc receptors include FcγRIIIa (CD16a), FcγRI (CD64), FcγRIIa(CD32), and FcαRI (CD89).

Antibody-dependent cell-mediated cytotoxicity (ADCC) is an immunemechanism leading to the lysis of antibody-coated target cells by immuneeffector cells. The target cells are cells to which antibodies orderivatives thereof comprising an Fc region specifically bind, generallyvia the protein part that is N-terminal to the Fc region. As usedherein, the term “reduced ADCC” is defined as either a reduction in thenumber of target cells that are lysed in a given time, at a givenconcentration of antibody in the medium surrounding the target cells, bythe mechanism of ADCC defined above, and/or an increase in theconcentration of antibody in the medium surrounding the target cells,required to achieve the lysis of a given number of target cells in agiven time, by the mechanism of ADCC. The reduction in ADCC is relativeto the ADCC mediated by the same antibody produced by the same type ofhost cells, using the same standard production, purification,formulation and storage methods (which are known to those skilled in theart), but that has not been engineered. For example the reduction inADCC mediated by an antibody comprising in its Fc domain an amino acidsubstitution that reduces ADCC, is relative to the ADCC mediated by thesame antibody without this amino acid substitution in the Fc domain.Suitable assays to measure ADCC are well known in the art (see e.g. PCTpublication no. WO 2006/082515 or PCT publication no. WO 2012/130831).

An “effective amount” of an agent refers to the amount that is necessaryto result in a physiological change in the cell or tissue to which it isadministered.

A “therapeutically effective amount” of an agent, e.g. a pharmaceuticalcomposition, refers to an amount effective, at dosages and for periodsof time necessary, to achieve the desired therapeutic or prophylacticresult. A therapeutically effective amount of an agent for exampleeliminates, decreases, delays, minimizes or prevents adverse effects ofa disease.

An “individual” or “subject” is a mammal. Mammals include, but are notlimited to, domesticated animals (e.g. cows, sheep, cats, dogs, andhorses), primates (e.g. humans and non-human primates such as monkeys),rabbits, and rodents (e.g. mice and rats). Particularly, the individualor subject is a human.

The term “pharmaceutical composition” refers to a preparation which isin such form as to permit the biological activity of an activeingredient contained therein to be effective, and which contains noadditional components which are unacceptably toxic to a subject to whichthe formulation would be administered.

A “pharmaceutically acceptable carrier” refers to an ingredient in apharmaceutical composition, other than an active ingredient, which isnontoxic to a subject. A pharmaceutically acceptable carrier includes,but is not limited to, a buffer, excipient, stabilizer, or preservative.

As used herein, “treatment” (and grammatical variations thereof such as“treat” or “treating”) refers to clinical intervention in an attempt toalter the natural course of a disease in the individual being treated,and can be performed either for prophylaxis or during the course ofclinical pathology. Desirable effects of treatment include, but are notlimited to, preventing occurrence or recurrence of disease, alleviationof symptoms, diminishment of any direct or indirect pathologicalconsequences of the disease, preventing metastasis, decreasing the rateof disease progression, amelioration or palliation of the disease state,and remission or improved prognosis. In some embodiments, T cellactivating bispecific antigen binding molecules of the invention areused to delay development of a disease or to slow the progression of adisease.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, dosage, administration,combination therapy, contraindications and/or warnings concerning theuse of such therapeutic products.

DETAILED DESCRIPTION OF THE EMBODIMENTS

The invention provides a T cell activating bispecific antigen bindingmolecule with favorable properties for therapeutic application, inparticular with improved produceability (e.g. with respect to purity,yield). The amino acid substitutions in Fab molecules comprised in the Tcell activating bispecific antigen binding molecules of the inventionare particularly efficient in reducing mispairing of light chains withnon-matching heavy chains (Bence-Jones-type side products), which canoccur in the production of Fab-based bi-/multispecific antigen bindingmolecules with a VH/VL exchange in one (or more, in case of moleculescomprising more than two antigen-binding Fab molecules) of their bindingarms (see also PCT application no. PCT/EP2015/057165, particularly theexamples therein, incorporated herein by reference in its entirety).

In a first aspect the invention provides a T cell activating bispecificantigen binding molecule comprising

-   -   (a) a first Fab molecule which specifically binds to a first        antigen    -   (b) a second Fab molecule which specifically binds to a second        antigen, and wherein the variable domains VL and VH of the Fab        light chain and the Fab heavy chain are replaced by each other,        wherein the first antigen is an activating T cell antigen and        the second antigen is a target cell antigen, or the first        antigen is a target cell antigen and the second antigen is an        activating T cell antigen; and wherein    -   i) in the constant domain CL of the first Fab molecule under a)        the amino acid at position 124 is substituted by a positively        charged amino acid (numbering according to Kabat), and wherein        in the constant domain CH1 of the first Fab molecule under a)        the amino acid at position 147 or the amino acid at position 213        is substituted by a negatively charged amino acid (numbering        according to Kabat EU index); or    -   ii) in the constant domain CL of the second Fab molecule        under b) the amino acid at position 124 is substituted by a        positively charged amino acid (numbering according to Kabat),        and wherein in the constant domain CH1 of the second Fab        molecule under b) the amino acid at position 147 or the amino        acid at position 213 is substituted by a negatively charged        amino acid (numbering according to Kabat EU index).

According to the invention, the T cell activating bispecific antigenbinding molecule does not comprise both modifications mentioned under i)and ii). The constant domains CL and CH1 of the second Fab molecule arenot replaced by each other (i.e. remain unexchanged).

In one embodiment of the T cell activating bispecific antigen bindingmolecule according to the invention, in the constant domain CL of thefirst Fab molecule under a) the amino acid at position 124 issubstituted independently by lysine (K), arginine (R) or histidine (H)(numbering according to Kabat) (in one preferred embodimentindependently by lysine (K) or arginine (R)), and in the constant domainCH1 of the first Fab molecule under a) the amino acid at position 147 orthe amino acid at position 213 is substituted independently by glutamicacid (E), or aspartic acid (D) (numbering according to Kabat EU index).

In a further embodiment, in the constant domain CL of the first Fabmolecule under a) the amino acid at position 124 is substitutedindependently by lysine (K), arginine (R) or histidine (H) (numberingaccording to Kabat), and in the constant domain CH1 of the first Fabmolecule under a) the amino acid at position 147 is substitutedindependently by glutamic acid (E), or aspartic acid (D) (numberingaccording to Kabat EU index).

In a particular embodiment, in the constant domain CL of the first Fabmolecule under a) the amino acid at position 124 is substitutedindependently by lysine (K), arginine (R) or histidine (H) (numberingaccording to Kabat) (in one preferred embodiment independently by lysine(K) or arginine (R)) and the amino acid at position 123 is substitutedindependently by lysine (K), arginine (R) or histidine (H) (numberingaccording to Kabat) (in one preferred embodiment independently by lysine(K) or arginine (R)), and in the constant domain CH1 of the first Fabmolecule under a) the amino acid at position 147 is substitutedindependently by glutamic acid (E), or aspartic acid (D) (numberingaccording to Kabat EU index) and the amino acid at position 213 issubstituted independently by glutamic acid (E), or aspartic acid (D)(numbering according to Kabat EU index).

In a more particular embodiment, in the constant domain CL of the firstFab molecule under a) the amino acid at position 124 is substituted bylysine (K) (numbering according to Kabat) and the amino acid at position123 is substituted by lysine (K) or arginine (R) (numbering according toKabat), and in the constant domain CH1 of the first Fab molecule undera) the amino acid at position 147 is substituted by glutamic acid (E)(numbering according to Kabat EU index) and the amino acid at position213 is substituted by glutamic acid (E) (numbering according to Kabat EUindex).

In an even more particular embodiment, in the constant domain CL of thefirst Fab molecule under a) the amino acid at position 124 issubstituted by lysine (K) (numbering according to Kabat) and the aminoacid at position 123 is substituted by arginine (R) (numbering accordingto Kabat), and in the constant domain CH1 of the first Fab moleculeunder a) the amino acid at position 147 is substituted by glutamic acid(E) (numbering according to Kabat EU index) and the amino acid atposition 213 is substituted by glutamic acid (E) (numbering according toKabat EU index).

In particular embodiments, the constant domain CL of the first Fabmolecule under a) is of kappa isotype.

Alternatively, the amino acid substitutions according to the aboveembodiments may be made in the constant domain CL and the constantdomain CH1 of the second Fab molecule under b) instead of in theconstant domain CL and the constant domain CH1 of the first Fab moleculeunder a). In particular such embodiments, the constant domain CL of thesecond Fab molecule under b) is of kappa isotype.

The T cell activating bispecific antigen binding molecule according tothe invention may further comprise a third Fab molecule whichspecifically binds to the first antigen. In particular embodiments, saidthird Fab molecule is identical to the first Fab molecule under a). Inthese embodiments, the amino acid substitutions according to the aboveembodiments will be made in the constant domain CL and the constantdomain CH1 of each of the first Fab molecule and the third Fab molecule.Alternatively, the amino acid substitutions according to the aboveembodiments may be made in the constant domain CL and the constantdomain CH1 of the second Fab molecule under b), but not in the constantdomain CL and the constant domain CH1 of the first Fab molecule and thethird Fab molecule.

In particular embodiments, the T cell activating bispecific antigenbinding molecule according to the invention further comprises an Fcdomain composed of a first and a second subunit capable of stableassociation.

T Cell Activating Bispecific Antigen Binding Molecule Formats

The components of the T cell activating bispecific antigen bindingmolecule can be fused to each other in a variety of configurations.Exemplary configurations are depicted in FIGS. 1A-1Z.

In particular embodiments, the T cell activating bispecific antigenbinding molecule comprises an Fc domain composed of a first and a secondsubunit capable of stable association.

In some embodiments, the second Fab molecule is fused at the C-terminusof the Fab heavy chain to the N-terminus of the first or the secondsubunit of the Fc domain.

In one such embodiment, the first Fab molecule is fused at theC-terminus of the Fab heavy chain to the N-terminus of the Fab heavychain of the second Fab molecule. In a specific such embodiment, the Tcell activating bispecific antigen binding molecule essentially consistsof the first and the second Fab molecule, the Fc domain composed of afirst and a second subunit, and optionally one or more peptide linkers,wherein the first Fab molecule is fused at the C-terminus of the Fabheavy chain to the N-terminus of the Fab heavy chain of the second Fabmolecule, and the second Fab molecule is fused at the C-terminus of theFab heavy chain to the N-terminus of the first or the second subunit ofthe Fc domain. Such a configuration is schematically depicted in FIGS.1G and 1K. Optionally, the Fab light chain of the first Fab molecule andthe Fab light chain of the second Fab molecule may additionally be fusedto each other.

In another such embodiment, the first Fab molecule is fused at theC-terminus of the Fab heavy chain to the N-terminus of the first orsecond subunit of the Fc domain. In a specific such embodiment, the Tcell activating bispecific antigen binding molecule essentially consistsof the first and the second Fab molecule, the Fc domain composed of afirst and a second subunit, and optionally one or more peptide linkers,wherein the first and the second Fab molecule are each fused at theC-terminus of the Fab heavy chain to the N-terminus of one of thesubunits of the Fc domain. Such a configuration is schematicallydepicted in FIGS. 1A and 1D. The first and the second Fab molecule maybe fused to the Fc domain directly or through a peptide linker. In aparticular embodiment the first and the second Fab molecule are eachfused to the Fc domain through an immunoglobulin hinge region. In aspecific embodiment, the immunoglobulin hinge region is a human IgG₁hinge region, particularly where the Fc domain is an IgG₁ Fc domain.

In other embodiments, the first Fab molecule is fused at the C-terminusof the Fab heavy chain to the N-terminus of the first or second subunitof the Fc domain.

In one such embodiment, the second Fab molecule is fused at theC-terminus of the Fab heavy chain to the N-terminus of the Fab heavychain of the first Fab molecule. In a specific such embodiment, the Tcell activating bispecific antigen binding molecule essentially consistsof the first and the second Fab molecule, the Fc domain composed of afirst and a second subunit, and optionally one or more peptide linkers,wherein the second Fab molecule is fused at the C-terminus of the Fabheavy chain to the N-terminus of the Fab heavy chain of the first Fabmolecule, and the first Fab molecule is fused at the C-terminus of theFab heavy chain to the N-terminus of the first or the second subunit ofthe Fc domain. Such a configuration is schematically depicted in FIGS.1H and 1L. Optionally, the Fab light chain of the first Fab molecule andthe Fab light chain of the second Fab molecule may additionally be fusedto each other.

The Fab molecules may be fused to the Fc domain or to each otherdirectly or through a peptide linker, comprising one or more aminoacids, typically about 2-20 amino acids. Peptide linkers are known inthe art and are described herein. Suitable, non-immunogenic peptidelinkers include, for example, (G₄S)_(n), (SG₄)_(n), (G₄S)_(n) orG₄(SG₄)_(n), peptide linkers. “n” is generally an integer from 1 to 10,typically from 2 to 4. In one embodiment said peptide linker has alength of at least 5 amino acids, in one embodiment a length of 5 to100, in a further embodiment of 10 to 50 amino acids. In one embodimentsaid peptide linker is (GxS)_(n) or (GxS)_(n)G_(m), with G=glycine,S=serine, and (x=3, n=3, 4, 5 or 6, and m=0, 1, 2 or 3) or (x=4, n=2, 3,4 or 5 and m=0, 1, 2 or 3), in one embodiment x=4 and n=2 or 3, in afurther embodiment x=4 and n=2. In one embodiment said peptide linker is(G₄S)₂. A particularly suitable peptide linker for fusing the Fab lightchains of the first and the second Fab molecule to each other is (G₄S)₂.An exemplary peptide linker suitable for connecting the Fab heavy chainsof the first and the second Fab fragments comprises the sequence(D)-(G₄S)₂ (SEQ ID NOs 11 and 12). Additionally, linkers may comprise (aportion of) an immunoglobulin hinge region. Particularly where a Fabmolecule is fused to the N-terminus of an Fc domain subunit, it may befused via an immunoglobulin hinge region or a portion thereof, with orwithout an additional peptide linker.

A T cell activating bispecific antigen binding molecule with a singleantigen binding moiety (such as a Fab molecule) capable of specificbinding to a target cell antigen (for example as shown in FIGS. 1A, 1D,1G, 1H, 1K, and 1L) is useful, particularly in cases whereinternalization of the target cell antigen is to be expected followingbinding of a high affinity antigen binding moiety. In such cases, thepresence of more than one antigen binding moiety specific for the targetcell antigen may enhance internalization of the target cell antigen,thereby reducing its availability.

In many other cases, however, it will be advantageous to have a T cellactivating bispecific antigen binding molecule comprising two or moreantigen binding moieties (such as Fab molecules) specific for a targetcell antigen (see examples shown in FIG. 1B, 1C, 1E, 1F, 1I, 1J. 1M or1N), for example to optimize targeting to the target site or to allowcrosslinking of target cell antigens.

Accordingly, in particular embodiments, the T cell activating bispecificantigen binding molecule of the invention further comprises a third Fabmolecule which specifically binds to the first antigen. The firstantigen preferably is the target cell antigen. In one embodiment, thethird Fab molecule is a conventional Fab molecule. In one embodiment,the third Fab molecule is identical to the first Fab molecule (i.e. thefirst and the third Fab molecule comprise the same heavy and light chainamino acid sequences and have the same arrangement of domains (i.e.conventional or crossover)). In a particular embodiment, the second Fabmolecule specifically binds to an activating T cell antigen,particularly CD3, and the first and third Fab molecule specifically bindto a target cell antigen.

In alternative embodiments, the T cell activating bispecific antigenbinding molecule of the invention further comprises a third Fab moleculewhich specifically binds to the second antigen. In these embodiments,the second antigen preferably is the target cell antigen. In one suchembodiment, the third Fab molecule is a crossover Fab molecule (a Fabmolecule wherein the variable domains VH and VL of the Fab heavy andlight chains are exchanged/replaced by each other). In one suchembodiment, the third Fab molecule is identical to the second Fabmolecule (i.e. the second and the third Fab molecule comprise the sameheavy and light chain amino acid sequences and have the same arrangementof domains (i.e. conventional or crossover)). In one such embodiment,the first Fab molecule specifically binds to an activating T cellantigen, particularly CD3, and the second and third Fab moleculespecifically bind to a target cell antigen.

In one embodiment, the third Fab molecule is fused at the C-terminus ofthe Fab heavy chain to the N-terminus of the first or second subunit ofthe Fc domain.

In a particular embodiment, the second and the third Fab molecule areeach fused at the C-terminus of the Fab heavy chain to the N-terminus ofone of the subunits of the Fc domain, and the first Fab molecule isfused at the C-terminus of the Fab heavy chain to the N-terminus of theFab heavy chain of the second Fab molecule. In a specific suchembodiment, the T cell activating bispecific antigen binding moleculeessentially consists of the first, the second and the third Fabmolecule, the Fc domain composed of a first and a second subunit, andoptionally one or more peptide linkers, wherein the first Fab moleculeis fused at the C-terminus of the Fab heavy chain to the N-terminus ofthe Fab heavy chain of the second Fab molecule, and the second Fabmolecule is fused at the C-terminus of the Fab heavy chain to theN-terminus of the first subunit of the Fc domain, and wherein the thirdFab molecule is fused at the C-terminus of the Fab heavy chain to theN-terminus of the second subunit of the Fc domain. Such a configurationis schematically depicted in FIGS. 1B and 1E (particular embodiments,wherein the third Fab molecule is a conventional Fab molecule andpreferably identical to the first Fab molecule), and FIGS. 1I and 1M(alternative embodiments, wherein the third Fab molecule is a crossoverFab molecule and preferably identical to the second Fab molecule). Thesecond and the third Fab molecule may be fused to the Fc domain directlyor through a peptide linker. In a particular embodiment the second andthe third Fab molecule are each fused to the Fc domain through animmunoglobulin hinge region. In a specific embodiment, theimmunoglobulin hinge region is a human IgG₁ hinge region, particularlywhere the Fc domain is an IgG₁ Fc domain. Optionally, the Fab lightchain of the first Fab molecule and the Fab light chain of the secondFab molecule may additionally be fused to each other.

In another embodiment, the first and the third Fab molecule are eachfused at the C-terminus of the Fab heavy chain to the N-terminus of oneof the subunits of the Fc domain, and the second Fab molecule is fusedat the C-terminus of the Fab heavy chain to the N-terminus of the Fabheavy chain of the first Fab molecule. In a specific such embodiment,the T cell activating bispecific antigen binding molecule essentiallyconsists of the first, the second and the third Fab molecule, the Fcdomain composed of a first and a second subunit, and optionally one ormore peptide linkers, wherein the second Fab molecule is fused at theC-terminus of the Fab heavy chain to the N-terminus of the Fab heavychain of the first Fab molecule, and the first Fab molecule is fused atthe C-terminus of the Fab heavy chain to the N-terminus of the firstsubunit of the Fc domain, and wherein the third Fab molecule is fused atthe C-terminus of the Fab heavy chain to the N-terminus of the secondsubunit of the Fc domain. Such a configuration is schematically depictedin FIGS. 1C and 1F (particular embodiments, wherein the third Fabmolecule is a conventional Fab molecule and preferably identical to thefirst Fab molecule) and in FIGS. 1J and 1N (alternative embodiments,wherein the third Fab molecule is a crossover Fab molecule andpreferably identical to the second Fab molecule). The first and thethird Fab molecule may be fused to the Fc domain directly or through apeptide linker. In a particular embodiment the first and the third Fabmolecule are each fused to the Fc domain through an immunoglobulin hingeregion. In a specific embodiment, the immunoglobulin hinge region is ahuman IgG₁ hinge region, particularly where the Fc domain is an IgG₁ Fcdomain. Optionally, the Fab light chain of the first Fab molecule andthe Fab light chain of the second Fab molecule may additionally be fusedto each other.

In configurations of the T cell activating bispecific antigen bindingmolecule wherein a Fab molecule is fused at the C-terminus of the Fabheavy chain to the N-terminus of each of the subunits of the Fc domainthrough an immunoglobulin hinge regions, the two Fab molecules, thehinge regions and the Fc domain essentially form an immunoglobulinmolecule. In a particular embodiment the immunoglobulin molecule is anIgG class immunoglobulin. In an even more particular embodiment theimmunoglobulin is an IgG₁ subclass immunoglobulin. In another embodimentthe immunoglobulin is an IgG₄ subclass immunoglobulin. In a furtherparticular embodiment the immunoglobulin is a human immunoglobulin. Inother embodiments the immunoglobulin is a chimeric immunoglobulin or ahumanized immunoglobulin.

In some of the T cell activating bispecific antigen binding molecule ofthe invention, the Fab light chain of the first Fab molecule and the Fablight chain of the second Fab molecule are fused to each other,optionally via a peptide linker. Depending on the configuration of thefirst and the second Fab molecule, the Fab light chain of the first Fabmolecule may be fused at its C-terminus to the N-terminus of the Fablight chain of the second Fab molecule, or the Fab light chain of thesecond Fab molecule may be fused at its C-terminus to the N-terminus ofthe Fab light chain of the first Fab molecule. Fusion of the Fab lightchains of the first and the second Fab molecule further reducesmispairing of unmatched Fab heavy and light chains, and also reduces thenumber of plasmids needed for expression of some of the T cellactivating bispecific antigen binding molecules of the invention.

In certain embodiments the T cell activating bispecific antigen bindingmolecule according to the invention comprises a polypeptide wherein theFab light chain variable region of the second Fab molecule shares acarboxy-terminal peptide bond with the Fab heavy chain constant regionof the second Fab molecule (i.e. the second Fab molecule comprises acrossover Fab heavy chain, wherein the heavy chain variable region isreplaced by a light chain variable region), which in turn shares acarboxy-terminal peptide bond with an Fc domain subunit(VL₍₂₎-CH1₍₂₎-CH2-CH3(-CH4)), and a polypeptide wherein the Fab heavychain of the first Fab molecule shares a carboxy-terminal peptide bondwith an Fc domain subunit (VH₍₁₎-CH1₍₁₎-CH2-CH3(-CH4)). In someembodiments the T cell activating bispecific antigen binding moleculefurther comprises a polypeptide wherein the Fab heavy chain variableregion of the second Fab molecule shares a carboxy-terminal peptide bondwith the Fab light chain constant region of the second Fab molecule(VH₍₂₎-CL₍₂₎) and the Fab light chain polypeptide of the first Fabmolecule (VL₍₁₎-CL₍₁₎). In certain embodiments the polypeptides arecovalently linked, e.g., by a disulfide bond.

In some embodiments, the T cell activating bispecific antigen bindingmolecule comprises a polypeptide wherein the Fab light chain variableregion of the second Fab molecule shares a carboxy-terminal peptide bondwith the Fab heavy chain constant region of the second Fab molecule(i.e. the second Fab molecule comprises a crossover Fab heavy chain,wherein the heavy chain variable region is replaced by a light chainvariable region), which in turn shares a carboxy-terminal peptide bondwith the Fab heavy chain of the first Fab molecule, which in turn sharesa carboxy-terminal peptide bond with an Fc domain subunit(VL₍₂₎-CH1₍₂₎-VH₍₁₎-CH1₍₁₎-CH2-CH3(-CH4)). In other embodiments, the Tcell activating bispecific antigen binding molecule comprises apolypeptide wherein the Fab heavy chain of the first Fab molecule sharesa carboxy-terminal peptide bond with the Fab light chain variable regionof the second Fab molecule which in turn shares a carboxy-terminalpeptide bond with the Fab heavy chain constant region of the second Fabmolecule (i.e. the second Fab molecule comprises a crossover Fab heavychain, wherein the heavy chain variable region is replaced by a lightchain variable region), which in turn shares a carboxy-terminal peptidebond with an Fc domain subunit(VH₍₁₎-CH1₍₁₎-VL₍₂₎-CH1₍₂₎-CH2-CH3(-CH4)).

In some of these embodiments the T cell activating bispecific antigenbinding molecule further comprises a crossover Fab light chainpolypeptide of the second Fab molecule, wherein the Fab heavy chainvariable region of the second Fab molecule shares a carboxy-terminalpeptide bond with the Fab light chain constant region of the second Fabmolecule (VH₍₂₎-CL₍₂₎), and the Fab light chain polypeptide of the firstFab molecule (VL₍₁₎-CL₍₁₎). In others of these embodiments the T cellactivating bispecific antigen binding molecule further comprises apolypeptide wherein the Fab light chain variable region of the secondFab molecule shares a carboxy-terminal peptide bond with the Fab heavychain constant region of the second Fab molecule which in turn shares acarboxy-terminal peptide bond with the Fab light chain polypeptide ofthe first Fab molecule (VL₍₂₎-CH1₍₂₎-VL₍₁₎-CL₍₁₎), or a polypeptidewherein the Fab light chain polypeptide of the first Fab molecule sharesa carboxy-terminal peptide bond with the Fab heavy chain variable regionof the second Fab molecule which in turn shares a carboxy-terminalpeptide bond with the Fab light chain constant region of the second Fabmolecule (VL₍₁₎-CL₍₁₎-VH₍₂₎-CL₍₂₎), as appropriate.

The T cell activating bispecific antigen binding molecule according tothese embodiments may further comprise (i) an Fc domain subunitpolypeptide (CH2-CH3(-CH4)), or (ii) a polypeptide wherein the Fab heavychain of a third Fab molecule shares a carboxy-terminal peptide bondwith an Fc domain subunit (VH₍₃₎-CH1₍₃₎-CH2-CH3(-CH4)) and the Fab lightchain polypeptide of a third Fab molecule (VL₍₃₎-CL₍₃₎). In certainembodiments the polypeptides are covalently linked, e.g., by a disulfidebond.

In some embodiments, the first Fab molecule is fused at the C-terminusof the Fab heavy chain to the N-terminus of the Fab heavy chain of thesecond Fab molecule. In certain such embodiments, the T cell activatingbispecific antigen binding molecule does not comprise an Fc domain. Incertain embodiments, the T cell activating bispecific antigen bindingmolecule essentially consists of the first and the second Fab molecule,and optionally one or more peptide linkers, wherein the first Fabmolecule is fused at the C-terminus of the Fab heavy chain to theN-terminus of the Fab heavy chain of the second Fab molecule. Such aconfiguration is schematically depicted in FIGS. 1O and 1S.

In other embodiments, the second Fab molecule is fused at the C-terminusof the Fab heavy chain to the N-terminus of the Fab heavy chain of thefirst Fab molecule. In certain such embodiments, the T cell activatingbispecific antigen binding molecule does not comprise an Fc domain. Incertain embodiments, the T cell activating bispecific antigen bindingmolecule essentially consists of the first and the second Fab molecule,and optionally one or more peptide linkers, wherein the second Fabmolecule is fused at the C-terminus of the Fab heavy chain to theN-terminus of the Fab heavy chain of the first Fab molecule. Such aconfiguration is schematically depicted in FIGS. 1P and 1T.

In some embodiments, the first Fab molecule is fused at the C-terminusof the Fab heavy chain to the N-terminus of the Fab heavy chain of thesecond Fab molecule, and the T cell activating bispecific antigenbinding molecule further comprises a third Fab molecule, wherein saidthird Fab molecule is fused at the C-terminus of the Fab heavy chain tothe N-terminus of the Fab heavy chain of the first Fab molecule. Inparticular such embodiments, said third Fab molecule is a conventionalFab molecule. In other such embodiments, said third Fab molecule is acrossover Fab molecule as described herein, i.e. a Fab molecule whereinthe variable domains VH and VL of the Fab heavy and light chains areexchanged/replaced by each other. In certain such embodiments, the Tcell activating bispecific antigen binding molecule essentially consistsof the first, the second and the third Fab molecule, and optionally oneor more peptide linkers, wherein the first Fab molecule is fused at theC-terminus of the Fab heavy chain to the N-terminus of the Fab heavychain of the second Fab molecule, and the third Fab molecule is fused atthe C-terminus of the Fab heavy chain to the N-terminus of the Fab heavychain of the first Fab molecule. Such a configuration is schematicallydepicted in FIGS. 1Q and 1U (particular embodiments, wherein the thirdFab molecule is a conventional Fab molecule and preferably identical tothe first Fab molecule).

In some embodiments, the first Fab molecule is fused at the C-terminusof the Fab heavy chain to the N-terminus of the Fab heavy chain of thesecond Fab molecule, and the T cell activating bispecific antigenbinding molecule further comprises a third Fab molecule, wherein saidthird Fab molecule is fused at the N-terminus of the Fab heavy chain tothe C-terminus of the Fab heavy chain of the second Fab molecule. Inparticular such embodiments, said third Fab molecule is a crossover Fabmolecule as described herein, i.e. a Fab molecule wherein the variabledomains VH and VL of the Fab heavy and light chains areexchanged/replaced by each other. In other such embodiments, said thirdFab molecule is a conventional Fab molecule. In certain suchembodiments, the T cell activating bispecific antigen binding moleculeessentially consists of the first, the second and the third Fabmolecule, and optionally one or more peptide linkers, wherein the firstFab molecule is fused at the C-terminus of the Fab heavy chain to theN-terminus of the Fab heavy chain of the second Fab molecule, and thethird Fab molecule is fused at the N-terminus of the Fab heavy chain tothe C-terminus of the Fab heavy chain of the second Fab molecule. Such aconfiguration is schematically depicted in FIGS. 1W and 1Y (particularembodiments, wherein the third Fab molecule is a crossover Fab moleculeand preferably identical to the second Fab molecule).

In some embodiments, the second Fab molecule is fused at the C-terminusof the Fab heavy chain to the N-terminus of the Fab heavy chain of thefirst Fab molecule, and the T cell activating bispecific antigen bindingmolecule further comprises a third Fab molecule, wherein said third Fabmolecule is fused at the N-terminus of the Fab heavy chain to theC-terminus of the Fab heavy chain of the first Fab molecule. Inparticular such embodiments, said third Fab molecule is a conventionalFab molecule. In other such embodiments, said third Fab molecule is acrossover Fab molecule as described herein, i.e. a Fab molecule whereinthe variable domains VH and VL of the Fab heavy and light chains areexchanged/replaced by each other. In certain such embodiments, the Tcell activating bispecific antigen binding molecule essentially consistsof the first, the second and the third Fab molecule, and optionally oneor more peptide linkers, wherein the second Fab molecule is fused at theC-terminus of the Fab heavy chain to the N-terminus of the Fab heavychain of the first Fab molecule, and the third Fab molecule is fused atthe N-terminus of the Fab heavy chain to the C-terminus of the Fab heavychain of the first Fab molecule. Such a configuration is schematicallydepicted in FIGS. 1R and 1V (particular embodiments, wherein the thirdFab molecule is a conventional Fab molecule and preferably identical tothe first Fab molecule).

In some embodiments, the second Fab molecule is fused at the C-terminusof the Fab heavy chain to the N-terminus of the Fab heavy chain of thefirst Fab molecule, and the T cell activating bispecific antigen bindingmolecule further comprises a third Fab molecule, wherein said third Fabmolecule is fused at the C-terminus of the Fab heavy chain to theN-terminus of the Fab heavy chain of the second Fab molecule. Inparticular such embodiments, said third Fab molecule is a crossover Fabmolecule as described herein, i.e. a Fab molecule wherein the variabledomains VH and VL of the Fab heavy and light chains areexchanged/replaced by each other. In other such embodiments, said thirdFab molecule is a conventional Fab molecule. In certain suchembodiments, the T cell activating bispecific antigen binding moleculeessentially consists of the first, the second and the third Fabmolecule, and optionally one or more peptide linkers, wherein the secondFab molecule is fused at the C-terminus of the Fab heavy chain to theN-terminus of the Fab heavy chain of the first Fab molecule, and thethird Fab molecule is fused at the C-terminus of the Fab heavy chain tothe N-terminus of the Fab heavy chain of the second Fab molecule. Such aconfiguration is schematically depicted in FIGS. 1X and 1Z (particularembodiments, wherein the third Fab molecule is a crossover Fab moleculeand preferably identical to the first Fab molecule).

In certain embodiments the T cell activating bispecific antigen bindingmolecule according to the invention comprises a polypeptide wherein theFab heavy chain of the first Fab molecule shares a carboxy-terminalpeptide bond with the Fab light chain variable region of the second Fabmolecule, which in turn shares a carboxy-terminal peptide bond with theFab heavy chain constant region of the second Fab molecule (i.e. thesecond Fab molecule comprises a crossover Fab heavy chain, wherein theheavy chain variable region is replaced by a light chain variableregion) (VH₍₁₎-CH1₍₁₎-VL₍₂₎-CH1₍₂₎). In some embodiments the T cellactivating bispecific antigen binding molecule further comprises apolypeptide wherein the Fab heavy chain variable region of the secondFab molecule shares a carboxy-terminal peptide bond with the Fab lightchain constant region of the second Fab molecule (VH₍₂₎-CL₍₂₎) and theFab light chain polypeptide of the first Fab molecule (VL₍₁₎-CL₍₁₎).

In certain embodiments the T cell activating bispecific antigen bindingmolecule according to the invention comprises a polypeptide wherein theFab light chain variable region of the second Fab molecule shares acarboxy-terminal peptide bond with the Fab heavy chain constant regionof the second Fab molecule (i.e. the second Fab molecule comprises acrossover Fab heavy chain, wherein the heavy chain variable region isreplaced by a light chain variable region), which in turn shares acarboxy-terminal peptide bond with the Fab heavy chain of the first Fabmolecule (VL₍₂₎-CH1₍₂₎-VH₍₁₎-CH1₍₁₎). In some embodiments the T cellactivating bispecific antigen binding molecule further comprises apolypeptide wherein the Fab heavy chain variable region of the secondFab molecule shares a carboxy-terminal peptide bond with the Fab lightchain constant region of the second Fab molecule (VH₍₂₎-CL₍₂₎) and theFab light chain polypeptide of the first Fab molecule (VL₍₁₎-CL₍₁₎).

In certain embodiments the T cell activating bispecific antigen bindingmolecule according to the invention comprises a polypeptide wherein theFab heavy chain of a third Fab molecule shares a carboxy-terminalpeptide bond with the Fab heavy chain of the first Fab molecule, whichin turn shares a carboxy-terminal peptide bond with the Fab light chainvariable region of the second Fab molecule, which in turn shares acarboxy-terminal peptide bond with the Fab heavy chain constant regionof the second Fab molecule (i.e. the second Fab molecule comprises acrossover Fab heavy chain, wherein the heavy chain variable region isreplaced by a light chain variable region)(VH₍₃₎-CH1₍₃₎-VH₍₁₎-CH1₍₁₎-VL₍₂₎-CH1₍₂₎). In some embodiments the T cellactivating bispecific antigen binding molecule further comprises apolypeptide wherein the Fab heavy chain variable region of the secondFab molecule shares a carboxy-terminal peptide bond with the Fab lightchain constant region of the second Fab molecule (VH₍₂₎-CL₍₂₎) and theFab light chain polypeptide of the first Fab molecule (VL₍₁₎-CL₍₁₎). Insome embodiments the T cell activating bispecific antigen bindingmolecule further comprises the Fab light chain polypeptide of a thirdFab molecule (VL₍₃₎-CL₍₃₎).

In certain embodiments the T cell activating bispecific antigen bindingmolecule according to the invention comprises a polypeptide wherein theFab light chain variable region of the second Fab molecule shares acarboxy-terminal peptide bond with the Fab heavy chain constant regionof the second Fab molecule (i.e. the second Fab molecule comprises acrossover Fab heavy chain, wherein the heavy chain variable region isreplaced by a light chain variable region), which in turn shares acarboxy-terminal peptide bond with the Fab heavy chain of the first Fabmolecule, which in turn shares a carboxy-terminal peptide bond with theFab heavy chain of a third Fab molecule(VL₍₂₎-CH1₍₂₎-VH₍₁₎-CH1₍₁₎-VH₍₃₎-CH1₍₃₎). In some embodiments the T cellactivating bispecific antigen binding molecule further comprises apolypeptide wherein the Fab heavy chain variable region of the secondFab molecule shares a carboxy-terminal peptide bond with the Fab lightchain constant region of the second Fab molecule (VH₍₂₎-CL₍₂₎) and theFab light chain polypeptide of the first Fab molecule (VL₍₁₎-CL₍₁₎). Insome embodiments the T cell activating bispecific antigen bindingmolecule further comprises the Fab light chain polypeptide of a thirdFab molecule (VL₍₃₎-CL₍₃₎).

In certain embodiments the T cell activating bispecific antigen bindingmolecule according to the invention comprises a polypeptide wherein theFab heavy chain of the first Fab molecule shares a carboxy-terminalpeptide bond with the Fab light chain variable region of the second Fabmolecule, which in turn shares a carboxy-terminal peptide bond with theFab heavy chain constant region of the second Fab molecule (i.e. thesecond Fab molecule comprises a crossover Fab heavy chain, wherein theheavy chain variable region is replaced by a light chain variableregion), which in turn shares a carboxy-terminal peptide bond with theFab light chain variable region of a third Fab molecule, which in turnshares a carboxy-terminal peptide bond with the Fab heavy chain constantregion of a third Fab molecule (i.e. the third Fab molecule comprises acrossover Fab heavy chain, wherein the heavy chain variable region isreplaced by a light chain variable region)(VH₍₁₎-CH1₍₁₎-VL₍₂₎-CH1₍₂₎-VL₍₃₎-CH1₍₃₎). In some embodiments the T cellactivating bispecific antigen binding molecule further comprises apolypeptide wherein the Fab heavy chain variable region of the secondFab molecule shares a carboxy-terminal peptide bond with the Fab lightchain constant region of the second Fab molecule (VH₍₂₎-CL₍₂₎) and theFab light chain polypeptide of the first Fab molecule (VL₍₁₎-CL₍₁₎). Insome embodiments the T cell activating bispecific antigen bindingmolecule further comprises a polypeptide wherein the Fab heavy chainvariable region of a third Fab molecule shares a carboxy-terminalpeptide bond with the Fab light chain constant region of a third Fabmolecule (VH₍₃₎-CL₍₃₎). In certain embodiments the T cell activatingbispecific antigen binding molecule according to the invention comprisesa polypeptide wherein the Fab light chain variable region of a third Fabmolecule shares a carboxy-terminal peptide bond with the Fab heavy chainconstant region of a third Fab molecule (i.e. the third Fab moleculecomprises a crossover Fab heavy chain, wherein the heavy chain variableregion is replaced by a light chain variable region), which in turnshares a carboxy-terminal peptide bond with the Fab light chain variableregion of the second Fab molecule, which in turn shares acarboxy-terminal peptide bond with the Fab heavy chain constant regionof the second Fab molecule (i.e. the second Fab molecule comprises acrossover Fab heavy chain, wherein the heavy chain variable region isreplaced by a light chain variable region), which in turn shares acarboxy-terminal peptide bond with the Fab heavy chain of the first Fabmolecule (VL₍₃₎-CH1₍₃₎-VL₍₂₎-CH1₍₂₎-VH₍₁₎-CH1₍₁₎). In some embodimentsthe T cell activating bispecific antigen binding molecule furthercomprises a polypeptide wherein the Fab heavy chain variable region ofthe second Fab molecule shares a carboxy-terminal peptide bond with theFab light chain constant region of the second Fab molecule (VH₍₂₎-CL₍₂₎)and the Fab light chain polypeptide of the first Fab molecule(VL₍₁₎-CL₍₁₎). In some embodiments the T cell activating bispecificantigen binding molecule further comprises a polypeptide wherein the Fabheavy chain variable region of a third Fab molecule shares acarboxy-terminal peptide bond with the Fab light chain constant regionof a third Fab molecule (VH₍₃₎-CL₍₃₎).

According to any of the above embodiments, components of the T cellactivating bispecific antigen binding molecule (e.g. Fab molecules, Fcdomain) may be fused directly or through various linkers, particularlypeptide linkers comprising one or more amino acids, typically about 2-20amino acids, that are described herein or are known in the art.Suitable, non-immunogenic peptide linkers include, for example,(G₄S)_(n), (SG₄)_(n), (G₄S)_(n) or G₄(SG₄)_(n) peptide linkers, whereinn is generally an integer from 1 to 10, typically from 2 to 4.

Fc Domain

The Fc domain of the T cell activating bispecific antigen bindingmolecule consists of a pair of polypeptide chains comprising heavy chaindomains of an immunoglobulin molecule. For example, the Fc domain of animmunoglobulin G (IgG) molecule is a dimer, each subunit of whichcomprises the CH2 and CH3 IgG heavy chain constant domains. The twosubunits of the Fc domain are capable of stable association with eachother. In one embodiment the T cell activating bispecific antigenbinding molecule of the invention comprises not more than one Fc domain.

In one embodiment according the invention the Fc domain of the T cellactivating bispecific antigen binding molecule is an IgG Fc domain. In aparticular embodiment the Fc domain is an IgG₁ Fc domain. In anotherembodiment the Fc domain is an IgG₄ Fc domain. In a more specificembodiment, the Fc domain is an IgG₄ Fc domain comprising an amino acidsubstitution at position 5228 (Kabat numbering), particularly the aminoacid substitution S228P. This amino acid substitution reduces in vivoFab arm exchange of IgG₄ antibodies (see Stubenrauch et al., DrugMetabolism and Disposition 38, 84-91 (2010)). In a further particularembodiment the Fc domain is human. An exemplary sequence of a human IgG₁Fc region is given in SEQ ID NO: 13.

Fc Domain Modifications Promoting Heterodimerization

T cell activating bispecific antigen binding molecules according to theinvention comprise different Fab molecules, fused to one or the other ofthe two subunits of the Fc domain, thus the two subunits of the Fcdomain are typically comprised in two non-identical polypeptide chains.Recombinant co-expression of these polypeptides and subsequentdimerization leads to several possible combinations of the twopolypeptides. To improve the yield and purity of T cell activatingbispecific antigen binding molecules in recombinant production, it willthus be advantageous to introduce in the Fc domain of the T cellactivating bispecific antigen binding molecule a modification promotingthe association of the desired polypeptides.

Accordingly, in particular embodiments the Fc domain of the T cellactivating bispecific antigen binding molecule according to theinvention comprises a modification promoting the association of thefirst and the second subunit of the Fc domain. The site of mostextensive protein-protein interaction between the two subunits of ahuman IgG Fc domain is in the CH3 domain of the Fc domain. Thus, in oneembodiment said modification is in the CH3 domain of the Fc domain.

There exist several approaches for modifications in the CH3 domain ofthe Fc domain in order to enforce heterodimerization, which are welldescribed e.g. in WO 96/27011, WO 98/050431, EP 1870459, WO 2007/110205,WO 2007/147901, WO 2009/089004, WO 2010/129304, WO 2011/90754, WO2011/143545, WO 2012058768, WO 2013157954, WO 2013096291.

Typically, in all such approaches the CH3 domain of the first subunit ofthe Fc domain and the CH3 domain of the second subunit of the Fc domainare both engineered in a complementary manner so that each CH3 domain(or the heavy chain comprising it) can no longer homodimerize withitself but is forced to heterodimerize with the complementarilyengineered other CH3 domain (so that the first and second CH3 domainheterodimerize and no homdimers between the two first or the two secondCH3 domains are formed). These different approaches for improved heavychain heterodimerization are contemplated as different alternatives incombination with the heavy-light chain modifications (VH and VLexchange/replacement in one binding arm and the introduction ofsubstitutions of charged amino acids with opposite charges in the CH1/CLinterface) in the T cell activating bispecific antigen binding moleculeaccording to the invention which reduce light chain mispairing and BenceJones-type side products.

In a specific embodiment said modification promoting the association ofthe first and the second subunit of the Fc domain is a so-called“knob-into-hole” modification, comprising a “knob” modification in oneof the two subunits of the Fc domain and a “hole” modification in theother one of the two subunits of the Fc domain.

The knob-into-hole technology is described e.g. in U.S. Pat. Nos.5,731,168; 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996) andCarter, J Immunol Meth 248, 7-15 (2001). Generally, the method involvesintroducing a protuberance (“knob”) at the interface of a firstpolypeptide and a corresponding cavity (“hole”) in the interface of asecond polypeptide, such that the protuberance can be positioned in thecavity so as to promote heterodimer formation and hinder homodimerformation. Protuberances are constructed by replacing small amino acidside chains from the interface of the first polypeptide with larger sidechains (e.g. tyrosine or tryptophan). Compensatory cavities of identicalor similar size to the protuberances are created in the interface of thesecond polypeptide by replacing large amino acid side chains withsmaller ones (e.g. alanine or threonine).

Accordingly, in a particular embodiment, in the CH3 domain of the firstsubunit of the Fc domain of the T cell activating bispecific antigenbinding molecule an amino acid residue is replaced with an amino acidresidue having a larger side chain volume, thereby generating aprotuberance within the CH3 domain of the first subunit which ispositionable in a cavity within the CH3 domain of the second subunit,and in the CH3 domain of the second subunit of the Fc domain an aminoacid residue is replaced with an amino acid residue having a smallerside chain volume, thereby generating a cavity within the CH3 domain ofthe second subunit within which the protuberance within the CH3 domainof the first subunit is positionable.

Preferably said amino acid residue having a larger side chain volume isselected from the group consisting of arginine (R), phenylalanine (F),tyrosine (Y), and tryptophan (W).

Preferably said amino acid residue having a smaller side chain volume isselected from the group consisting of alanine (A), serine (S), threonine(T), and valine (V).

The protuberance and cavity can be made by altering the nucleic acidencoding the polypeptides, e.g. by site-specific mutagenesis, or bypeptide synthesis.

In a specific embodiment, in the CH3 domain of the first subunit of theFc domain (the “knobs” subunit) the threonine residue at position 366 isreplaced with a tryptophan residue (T366W), and in the CH3 domain of thesecond subunit of the Fc domain (the “hole” subunit) the tyrosineresidue at position 407 is replaced with a valine residue (Y407V). Inone embodiment, in the second subunit of the Fc domain additionally thethreonine residue at position 366 is replaced with a serine residue(T366S) and the leucine residue at position 368 is replaced with analanine residue (L368A) (numberings according to Kabat EU index).

In yet a further embodiment, in the first subunit of the Fc domainadditionally the serine residue at position 354 is replaced with acysteine residue (S354C) or the glutamic acid residue at position 356 isreplaced with a cysteine residue (E356C), and in the second subunit ofthe Fc domain additionally the tyrosine residue at position 349 isreplaced by a cysteine residue (Y349C) (numberings according to Kabat EUindex). Introduction of these two cysteine residues results in formationof a disulfide bridge between the two subunits of the Fc domain, furtherstabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)).

In a particular embodiment, the first subunit of the Fc domain comprisesamino acid substitutions S354C and T366W, and the second subunit of theFc domain comprises amino acid substitutions Y349C, T366S, L368A andY407V (numbering according to Kabat EU index).

In a particular embodiment the Fab molecule which specifically binds anactivating T cell antigen is fused (optionally via a Fab molecule whichspecifically binds to a target cell antigen) to the first subunit of theFc domain (comprising the “knob” modification). Without wishing to bebound by theory, fusion of the Fab molecule which specifically binds anactivating T cell antigen to the knob-containing subunit of the Fcdomain will (further) minimize the generation of antigen bindingmolecules comprising two Fab molecules which bind to an activating Tcell antigen (steric clash of two knob-containing polypeptides).

Other techniques of CH3-modification for enforcing theheterodimerization are contemplated as alternatives according to theinvention and are described e.g. in WO 96/27011, WO 98/050431, EP1870459, WO 2007/110205, WO 2007/147901, WO 2009/089004, WO 2010/129304,WO 2011/90754, WO 2011/143545, WO 2012/058768, WO 2013/157954, WO2013/096291.

In one embodiment the heterodimerization approach described in EP1870459 A1, is used alternatively. This approach is based on theintroduction of charged amino acids with opposite charges at specificamino acid positions in the CH3/CH3 domain interface between the twosubunits of the Fc domain. One preferred embodiment for the T cellactivating bispecific antigen binding molecule of the invention areamino acid mutations R409D; K370E in one of the two CH3 domains (of theFc domain) and amino acid mutations D399K; E357K in the other one of theCH3 domains of the Fc domain (numbering according to Kabat EU index).

In another embodiment the T cell activating bispecific antigen bindingmolecule of the invention comprises amino acid mutation T366W in the CH3domain of the first subunit of the Fc domain and amino acid mutationsT366S, L368A, Y407V in the CH3 domain of the second subunit of the Fcdomain, and additionally amino acid mutations R409D; K370E in the CH3domain of the first subunit of the Fc domain and amino acid mutationsD399K; E357K in the CH3 domain of the second subunit of the Fc domain(numberings according to Kabat EU index).

In another embodiment T cell activating bispecific antigen bindingmolecule of the invention comprises amino acid mutations S354C, T366W inthe CH3 domain of the first subunit of the Fc domain and amino acidmutations Y349C, T366S, L368A, Y407V in the CH3 domain of the secondsubunit of the Fc domain, or said T cell activating bispecific antigenbinding molecule comprises amino acid mutations Y349C, T366W in the CH3domain of the first subunit of the Fc domain and amino acid mutationsS354C, T366S, L368A, Y407V in the CH3 domains of the second subunit ofthe Fc domain and additionally amino acid mutations R409D; K370E in theCH3 domain of the first subunit of the Fc domain and amino acidmutations D399K; E357K in the CH3 domain of the second subunit of the Fcdomain (all numberings according to Kabat EU index).

In one embodiment the heterodimerization approach described in WO2013/157953 is used alternatively. In one embodiment a first CH3 domaincomprises amino acid mutation T366K and a second CH3 domain comprisesamino acid mutation L351D (numberings according to Kabat EU index). In afurther embodiment the first CH3 domain comprises further amino acidmutation L351K. In a further embodiment the second CH3 domain comprisesfurther an amino acid mutation selected from Y349E, Y349D and L368E(preferably L368E) (numberings according to Kabat EU index).

In one embodiment the heterodimerization approach described in WO2012/058768 is used alternatively. In one embodiment a first CH3 domaincomprises amino acid mutations L351Y, Y407A and a second CH3 domaincomprises amino acid mutations T366A, K409F. In a further embodiment thesecond CH3 domain comprises a further amino acid mutation at positionT411, D399, 5400, F405, N390, or K392, e.g. selected from a) T411N,T411R, T411Q, T411K, T411D, T411E or T411W, b) D399R, D399W, D399Y orD399K, c) S400E, S400D, S400R, or S400K, d) F4051, F405M, F405T, F405S,F405V or F405W, e) N390R, N390K or N390D, f) K392V, K392M, K392R, K392L,K392F or K392E (numberings according to Kabat EU index). In a furtherembodiment a first CH3 domain comprises amino acid mutations L351Y,Y407A and a second CH3 domain comprises amino acid mutations T366V,K409F. In a further embodiment a first CH3 domain comprises amino acidmutation Y407A and a second CH3 domain comprises amino acid mutationsT366A, K409F. In a further embodiment the second CH3 domain furthercomprises amino acid mutations K392E, T411E, D399R and S400R (numberingsaccording to Kabat EU index).

In one embodiment the heterodimerization approach described in WO2011/143545 is used alternatively, e.g. with the amino acid modificationat a position selected from the group consisting of 368 and 409(numbering according to Kabat EU index).

In one embodiment the heterodimerization approach described in WO2011/090762, which also uses the knobs-into-holes technology describedabove, is used alternatively. In one embodiment a first CH3 domaincomprises amino acid mutation T366W and a second CH3 domain comprisesamino acid mutation Y407A. In one embodiment a first CH3 domaincomprises amino acid mutation T366Y and a second CH3 domain comprisesamino acid mutation Y407T (numberings according to Kabat EU index).

In one embodiment the T cell activating bispecific antigen bindingmolecule or its Fc domain is of IgG₂ subclass and the heterodimerizationapproach described in WO 2010/129304 is used alternatively.

In an alternative embodiment a modification promoting association of thefirst and the second subunit of the Fc domain comprises a modificationmediating electrostatic steering effects, e.g. as described in PCTpublication WO 2009/089004. Generally, this method involves replacementof one or more amino acid residues at the interface of the two Fc domainsubunits by charged amino acid residues so that homodimer formationbecomes electrostatically unfavorable but heterodimerizationelectrostatically favorable. In one such embodiment a first CH3 domaincomprises amino acid substitution of K392 or N392 with a negativelycharged amino acid (e.g. glutamic acid (E), or aspartic acid (D),preferably K392D or N392D) and a second CH3 domain comprises amino acidsubstitution of D399, E356, D356, or E357 with a positively chargedamino acid (e.g. lysine (K) or arginine (R), preferably D399K, E356K,D356K, or E357K, and more preferably D399K and E356K). In a furtherembodiment the first CH3 domain further comprises amino acidsubstitution of K409 or R409 with a negatively charged amino acid (e.g.glutamic acid (E), or aspartic acid (D), preferably K409D or R409D). Ina further embodiment the first CH3 domain further or alternativelycomprises amino acid substitution of K439 and/or K370 with a negativelycharged amino acid (e.g. glutamic acid (E), or aspartic acid (D)) (allnumberings according to Kabat EU index).

In yet a further embodiment the heterodimerization approach described inWO 2007/147901 is used alternatively. In one embodiment a first CH3domain comprises amino acid mutations K253E, D282K, and K322D and asecond CH3 domain comprises amino acid mutations D239K, E240K, and K292D(numberings according to Kabat EU index).

In still another embodiment the heterodimerization approach described inWO 2007/110205 can be used alternatively.

In one embodiment, the first subunit of the Fc domain comprises aminoacid substitutions K392D and K409D, and the second subunit of the Fcdomain comprises amino acid substitutions D356K and D399K (numberingaccording to Kabat EU index).

Fc Domain Modifications Reducing Fc Receptor Binding and/or EffectorFunction

The Fc domain confers to the T cell activating bispecific antigenbinding molecule favorable pharmacokinetic properties, including a longserum half-life which contributes to good accumulation in the targettissue and a favorable tissue-blood distribution ratio. At the same timeit may, however, lead to undesirable targeting of the T cell activatingbispecific antigen binding molecule to cells expressing Fc receptorsrather than to the preferred antigen-bearing cells. Moreover, theco-activation of Fc receptor signaling pathways may lead to cytokinerelease which, in combination with the T cell activating properties andthe long half-life of the antigen binding molecule, results in excessiveactivation of cytokine receptors and severe side effects upon systemicadministration. Activation of (Fc receptor-bearing) immune cells otherthan T cells may even reduce efficacy of the T cell activatingbispecific antigen binding molecule due to the potential destruction ofT cells e.g. by NK cells.

Accordingly, in particular embodiments, the Fc domain of the T cellactivating bispecific antigen binding molecules according to theinvention exhibits reduced binding affinity to an Fc receptor and/orreduced effector function, as compared to a native IgG₁ Fc domain. Inone such embodiment the Fc domain (or the T cell activating bispecificantigen binding molecule comprising said Fc domain) exhibits less than50%, preferably less than 20%, more preferably less than 10% and mostpreferably less than 5% of the binding affinity to an Fc receptor, ascompared to a native IgG₁ Fc domain (or a T cell activating bispecificantigen binding molecule comprising a native IgG₁ Fc domain), and/orless than 50%, preferably less than 20%, more preferably less than 10%and most preferably less than 5% of the effector function, as comparedto a native IgG₁ Fc domain domain (or a T cell activating bispecificantigen binding molecule comprising a native IgG₁ Fc domain). In oneembodiment, the Fc domain domain (or the T cell activating bispecificantigen binding molecule comprising said Fc domain) does notsubstantially bind to an Fc receptor and/or induce effector function. Ina particular embodiment the Fc receptor is an Fcγ receptor. In oneembodiment the Fc receptor is a human Fc receptor. In one embodiment theFc receptor is an activating Fc receptor. In a specific embodiment theFc receptor is an activating human Fcγ receptor, more specifically humanFcγRIIIa, FcγRI or FcγRIIa, most specifically human FcγRIIIa. In oneembodiment the effector function is one or more selected from the groupof CDC, ADCC, ADCP, and cytokine secretion. In a particular embodimentthe effector function is ADCC. In one embodiment the Fc domain domainexhibits substantially similar binding affinity to neonatal Fc receptor(FcRn), as compared to a native IgG₁ Fc domain domain. Substantiallysimilar binding to FcRn is achieved when the Fc domain (or the T cellactivating bispecific antigen binding molecule comprising said Fcdomain) exhibits greater than about 70%, particularly greater than about80%, more particularly greater than about 90% of the binding affinity ofa native IgG₁ Fc domain (or the T cell activating bispecific antigenbinding molecule comprising a native IgG₁ Fc domain) to FcRn.

In certain embodiments the Fc domain is engineered to have reducedbinding affinity to an Fc receptor and/or reduced effector function, ascompared to a non-engineered Fc domain. In particular embodiments, theFc domain of the T cell activating bispecific antigen binding moleculecomprises one or more amino acid mutation that reduces the bindingaffinity of the Fc domain to an Fc receptor and/or effector function.Typically, the same one or more amino acid mutation is present in eachof the two subunits of the Fc domain. In one embodiment the amino acidmutation reduces the binding affinity of the Fc domain to an Fcreceptor. In one embodiment the amino acid mutation reduces the bindingaffinity of the Fc domain to an Fc receptor by at least 2-fold, at least5-fold, or at least 10-fold. In embodiments where there is more than oneamino acid mutation that reduces the binding affinity of the Fc domainto the Fc receptor, the combination of these amino acid mutations mayreduce the binding affinity of the Fc domain to an Fc receptor by atleast 10-fold, at least 20-fold, or even at least 50-fold. In oneembodiment the T cell activating bispecific antigen binding moleculecomprising an engineered Fc domain exhibits less than 20%, particularlyless than 10%, more particularly less than 5% of the binding affinity toan Fc receptor as compared to a T cell activating bispecific antigenbinding molecule comprising a non-engineered Fc domain. In a particularembodiment the Fc receptor is an Fcγ receptor. In some embodiments theFc receptor is a human Fc receptor. In some embodiments the Fc receptoris an activating Fc receptor. In a specific embodiment the Fc receptoris an activating human Fey receptor, more specifically human FcγRIIIa,FcγRI or FcγRIIa, most specifically human FcγRIIIa. Preferably, bindingto each of these receptors is reduced. In some embodiments bindingaffinity to a complement component, specifically binding affinity toC1q, is also reduced. In one embodiment binding affinity to neonatal Fcreceptor (FcRn) is not reduced. Substantially similar binding to FcRn,i.e. preservation of the binding affinity of the Fc domain to saidreceptor, is achieved when the Fc domain (or the T cell activatingbispecific antigen binding molecule comprising said Fc domain) exhibitsgreater than about 70% of the binding affinity of a non-engineered formof the Fc domain (or the T cell activating bispecific antigen bindingmolecule comprising said non-engineered form of the Fc domain) to FcRn.The Fc domain, or T cell activating bispecific antigen binding moleculesof the invention comprising said Fc domain, may exhibit greater thanabout 80% and even greater than about 90% of such affinity. In certainembodiments the Fc domain of the T cell activating bispecific antigenbinding molecule is engineered to have reduced effector function, ascompared to a non-engineered Fc domain. The reduced effector functioncan include, but is not limited to, one or more of the following:reduced complement dependent cytotoxicity (CDC), reducedantibody-dependent cell-mediated cytotoxicity (ADCC), reducedantibody-dependent cellular phagocytosis (ADCP), reduced cytokinesecretion, reduced immune complex-mediated antigen uptake byantigen-presenting cells, reduced binding to NK cells, reduced bindingto macrophages, reduced binding to monocytes, reduced binding topolymorphonuclear cells, reduced direct signaling inducing apoptosis,reduced crosslinking of target-bound antibodies, reduced dendritic cellmaturation, or reduced T cell priming. In one embodiment the reducedeffector function is one or more selected from the group of reduced CDC,reduced ADCC, reduced ADCP, and reduced cytokine secretion. In aparticular embodiment the reduced effector function is reduced ADCC. Inone embodiment the reduced ADCC is less than 20% of the ADCC induced bya non-engineered Fc domain (or a T cell activating bispecific antigenbinding molecule comprising a non-engineered Fc domain).

In one embodiment the amino acid mutation that reduces the bindingaffinity of the Fc domain to an Fc receptor and/or effector function isan amino acid substitution. In one embodiment the Fc domain comprises anamino acid substitution at a position selected from the group of E233,L234, L235, N297, P331 and P329 (numberings according to Kabat EUindex). In a more specific embodiment the Fc domain comprises an aminoacid substitution at a position selected from the group of L234, L235and P329 (numberings according to Kabat EU index). In some embodimentsthe Fc domain comprises the amino acid substitutions L234A and L235A(numberings according to Kabat EU index). In one such embodiment, the Fcdomain is an IgG₁ Fc domain, particularly a human IgG₁ Fc domain. In oneembodiment the Fc domain comprises an amino acid substitution atposition P329. In a more specific embodiment the amino acid substitutionis P329A or P329G, particularly P329G (numberings according to Kabat EUindex). In one embodiment the Fc domain comprises an amino acidsubstitution at position P329 and a further amino acid substitution at aposition selected from E233, L234, L235, N297 and P331 (numberingsaccording to Kabat EU index). In a more specific embodiment the furtheramino acid substitution is E233P, L234A, L235A, L235E, N297A, N297D orP331S. In particular embodiments the Fc domain comprises amino acidsubstitutions at positions P329, L234 and L235 (numberings according toKabat EU index). In more particular embodiments the Fc domain comprisesthe amino acid mutations L234A, L235A and P329G (“P329G LALA”). In onesuch embodiment, the Fc domain is an IgG₁ Fc domain, particularly ahuman IgG₁ Fc domain. The “P329G LALA” combination of amino acidsubstitutions almost completely abolishes Fcγ receptor (as well ascomplement) binding of a human IgG₁ Fc domain, as described in PCTpublication no. WO 2012/130831, incorporated herein by reference in itsentirety. WO 2012/130831 also describes methods of preparing such mutantFc domains and methods for determining its properties such as Fcreceptor binding or effector functions.

IgG₄ antibodies exhibit reduced binding affinity to Fc receptors andreduced effector functions as compared to IgG₁ antibodies. Hence, insome embodiments the Fc domain of the T cell activating bispecificantigen binding molecules of the invention is an IgG₄ Fc domain,particularly a human IgG₄ Fc domain. In one embodiment the IgG₄ Fcdomain comprises amino acid substitutions at position S228, specificallythe amino acid substitution S228P (numberings according to Kabat EUindex). To further reduce its binding affinity to an Fc receptor and/orits effector function, in one embodiment the IgG₄ Fc domain comprises anamino acid substitution at position L235, specifically the amino acidsubstitution L235E (numberings according to Kabat EU index). In anotherembodiment, the IgG₄ Fc domain comprises an amino acid substitution atposition P329, specifically the amino acid substitution P329G(numberings according to Kabat EU index). In a particular embodiment,the IgG₄ Fc domain comprises amino acid substitutions at positions S228,L235 and P329, specifically amino acid substitutions S228P, L235E andP329G (numberings according to Kabat EU index). Such IgG₄ Fc domainmutants and their Fcγ receptor binding properties are described in PCTpublication no. WO 2012/130831, incorporated herein by reference in itsentirety.

In a particular embodiment the Fc domain exhibiting reduced bindingaffinity to an Fc receptor and/or reduced effector function, as comparedto a native IgG₁ Fc domain, is a human IgG₁ Fc domain comprising theamino acid substitutions L234A, L235A and optionally P329G, or a humanIgG₄ Fc domain comprising the amino acid substitutions S228P, L235E andoptionally P329G (numberings according to Kabat EU index).

In certain embodiments N-glycosylation of the Fc domain has beeneliminated. In one such embodiment the Fc domain comprises an amino acidmutation at position N297, particularly an amino acid substitutionreplacing asparagine by alanine (N297A) or aspartic acid (N297D)(numberings according to Kabat EU index).

In addition to the Fc domains described hereinabove and in PCTpublication no. WO 2012/130831, Fc domains with reduced Fc receptorbinding and/or effector function also include those with substitution ofone or more of Fc domain residues 238, 265, 269, 270, 297, 327 and 329(U.S. Pat. No. 6,737,056) (numberings according to Kabat EU index). SuchFc mutants include Fc mutants with substitutions at two or more of aminoacid positions 265, 269, 270, 297 and 327, including the so-called“DANA” Fc mutant with substitution of residues 265 and 297 to alanine(U.S. Pat. No. 7,332,581).

Mutant Fc domains can be prepared by amino acid deletion, substitution,insertion or modification using genetic or chemical methods well knownin the art. Genetic methods may include site-specific mutagenesis of theencoding DNA sequence, PCR, gene synthesis, and the like. The correctnucleotide changes can be verified for example by sequencing.

Binding to Fc receptors can be easily determined e.g. by ELISA, or bySurface Plasmon Resonance (SPR) using standard instrumentation such as aBIAcore instrument (GE Healthcare), and Fc receptors such as may beobtained by recombinant expression. A suitable such binding assay isdescribed herein. Alternatively, binding affinity of Fc domains or cellactivating bispecific antigen binding molecules comprising an Fc domainfor Fc receptors may be evaluated using cell lines known to expressparticular Fc receptors, such as human NK cells expressing FcγIIIareceptor.

Effector function of an Fc domain, or a T cell activating bispecificantigen binding molecule comprising an Fc domain, can be measured bymethods known in the art. A suitable assay for measuring ADCC isdescribed herein. Other examples of in vitro assays to assess ADCCactivity of a molecule of interest are described in U.S. Pat. No.5,500,362; Hellstrom et al. Proc Natl Acad Sci USA 83, 7059-7063 (1986)and Hellstrom et al., Proc Natl Acad Sci USA 82, 1499-1502 (1985); U.S.Pat. No. 5,821,337; Bruggemann et al., J Exp Med 166, 1351-1361 (1987).Alternatively, non-radioactive assays methods may be employed (see, forexample, ACTI™ non-radioactive cytotoxicity assay for flow cytometry(CellTechnology, Inc. Mountain View, Calif.); and CytoTox 96®non-radioactive cytotoxicity assay (Promega, Madison, Wis.)). Usefuleffector cells for such assays include peripheral blood mononuclearcells (PBMC) and Natural Killer (NK) cells. Alternatively, oradditionally, ADCC activity of the molecule of interest may be assessedin vivo, e.g. in a animal model such as that disclosed in Clynes et al.,Proc Natl Acad Sci USA 95, 652-656 (1998).

In some embodiments, binding of the Fc domain to a complement component,specifically to C1q, is reduced. Accordingly, in some embodimentswherein the Fc domain is engineered to have reduced effector function,said reduced effector function includes reduced CDC. C1q binding assaysmay be carried out to determine whether the T cell activating bispecificantigen binding molecule is able to bind C1q and hence has CDC activity.See e.g., C1q and C3c binding ELISA in WO 2006/029879 and WO2005/100402. To assess complement activation, a CDC assay may beperformed (see, for example, Gazzano-Santoro et al., J Immunol Methods202, 163 (1996); Cragg et al., Blood 101, 1045-1052 (2003); and Craggand Glennie, Blood 103, 2738-2743 (2004)).

Antigen Binding Moieties

The antigen binding molecule of the invention is bispecific, i.e. itcomprises at least two antigen binding moieties capable of specificbinding to two distinct antigenic determinants. According to theinvention, the antigen binding moieties are Fab molecules (i.e. antigenbinding domains composed of a heavy and a light chain, each comprising avariable and a constant domain). In one embodiment said Fab moleculesare human. In another embodiment said Fab molecules are humanized. Inyet another embodiment said Fab molecules comprise human heavy and lightchain constant domains.

At least one of the antigen binding moieties is a crossover Fabmolecule. Such modification reduces mispairing of heavy and light chainsfrom different Fab molecules, thereby improving the yield and purity ofthe T cell activating bispecific antigen binding molecule of theinvention in recombinant production. In a particular crossover Fabmolecule useful for the T cell activating bispecific antigen bindingmolecule of the invention, the variable domains of the Fab light chainand the Fab heavy chain (VL and VH, respectively) are exchanged. Evenwith this domain exchange, however, the preparation of the T cellactivating bispecific antigen binding molecule may comprise certain sideproducts due to a so-called Bence Jones-type interaction betweenmispaired heavy and light chains (see Schaefer et al, PNAS, 108 (2011)11187-11191). To further reduce mispairing of heavy and light chainsfrom different Fab molecules and thus increase the purity and yield ofthe desired T cell activating bispecific antigen binding molecule,according to the present invention charged amino acids with oppositecharges are introduced at specific amino acid positions in the CH1 andCL domains of either the Fab molecule(s) specifically binding to atarget cell antigen, or the Fab molecule specifically binding to anactivating T cell antigen. Charge modifications are made either in theconventional Fab molecule(s) comprised in the T cell activatingbispecific antigen binding molecule (such as shown e.g. in FIG. 1A-1Cand 1G-1J), or in the crossover Fab molecule(s) comprised in the T cellactivating bispecific antigen binding molecule (such as shown e.g. inFIGS. 1D-1F and 1K-1N) (but not in both). In particular embodiments, thecharge modifications are made in the conventional Fab molecule(s)comprised in the T cell activating bispecific antigen binding molecule(which in particular embodiments specifically bind(s) to the target cellantigen).

In a particular embodiment according to the invention, the T cellactivating bispecific antigen binding molecule is capable ofsimultaneous binding to a target cell antigen, particularly a tumor cellantigen, and an activating T cell antigen, particularly CD3. In oneembodiment, the T cell activating bispecific antigen binding molecule iscapable of crosslinking a T cell and a target cell by simultaneousbinding to a target cell antigen and an activating T cell antigen. In aneven more particular embodiment, such simultaneous binding results inlysis of the target cell, particularly a tumor cell. In one embodiment,such simultaneous binding results in activation of the T cell. In otherembodiments, such simultaneous binding results in a cellular response ofa T lymphocyte, particularly a cytotoxic T lymphocyte, selected from thegroup of: proliferation, differentiation, cytokine secretion, cytotoxiceffector molecule release, cytotoxic activity, and expression ofactivation markers. In one embodiment, binding of the T cell activatingbispecific antigen binding molecule to the activating T cell antigen,particularly CD3, without simultaneous binding to the target cellantigen does not result in T cell activation.

In one embodiment, the T cell activating bispecific antigen bindingmolecule is capable of re-directing cytotoxic activity of a T cell to atarget cell. In a particular embodiment, said re-direction isindependent of MHC-mediated peptide antigen presentation by the targetcell and and/or specificity of the T cell.

Particularly, a T cell according to any of the embodiments of theinvention is a cytotoxic T cell. In some embodiments the T cell is aCD4⁺ or a CD8⁺ T cell, particularly a CD8⁺ T cell.

Activating T Cell Antigen Binding Fab Molecule

The T cell activating bispecific antigen binding molecule of theinvention comprises at least one Fab molecule which specifically bindsto an activating T cell antigen (also referred to herein as an“activating T cell antigen binding Fab molecule”). In a particularembodiment, the T cell activating bispecific antigen binding moleculecomprises not more than one Fab molecule (or other Fab molecule) capableof specific binding to an activating T cell antigen. In one embodimentthe T cell activating bispecific antigen binding molecule providesmonovalent binding to the activating T cell antigen.

In particular embodiments, the Fab molecule which specifically binds anactivating T cell antigen is a crossover Fab molecule as describedherein, i.e. a Fab molecule wherein the variable domains VH and VL ofthe Fab heavy and light chains are exchanged/replaced by each other. Insuch embodiments, the Fab molecule(s) which specifically binds a targetcell antigen is a conventional Fab molecule. In embodiments where thereis more than one Fab molecule which specifically binds to a target cellantigen comprised in the T cell activating bispecific antigen bindingmolecule, the Fab molecule which specifically binds to an activating Tcell antigen preferably is a crossover Fab molecule and the Fabmolecules which specifically bind to a target cell antigen areconventional Fab molecules.

In alternative embodiments, the Fab molecule which specifically binds anactivating T cell antigen is a conventional Fab molecule. In suchembodiments, the Fab molecule(s) which specifically binds a target cellantigen is a crossover Fab molecule as described herein, i.e. a Fabmolecule wherein the variable domains VH and VL of the Fab heavy andlight chains are exchanged/replaced by each other.

In a particular embodiment the activating T cell antigen is CD3,particularly human CD3 (SEQ ID NO: 1) or cynomolgus CD3 (SEQ ID NO: 2),most particularly human CD3. In a particular embodiment the activating Tcell antigen binding Fab molecule is cross-reactive for (i.e.specifically binds to) human and cynomolgus CD3. In some embodiments,the activating T cell antigen is the epsilon subunit of CD3 (CD3epsilon).

In some embodiments, the activating T cell antigen binding Fab moleculespecifically binds to CD3, particularly CD3 epsilon, and comprises atleast one heavy chain complementarity determining region (CDR) selectedfrom the group consisting of SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6and at least one light chain CDR selected from the group of SEQ ID NO:8, SEQ ID NO: 9, SEQ ID NO: 10.

In one embodiment the CD3 binding Fab molecule comprises a heavy chainvariable region comprising the heavy chain CDR1 of SEQ ID NO: 4, theheavy chain CDR2 of SEQ ID NO: 5, the heavy chain CDR3 of SEQ ID NO: 6,and a light chain variable region comprising the light chain CDR1 of SEQID NO: 8, the light chain CDR2 of SEQ ID NO: 9, and the light chain CDR3of SEQ ID NO: 10.

In another embodiment the CD3 binding Fab molecule comprises a heavychain variable region comprising the heavy chain CDR1 of SEQ ID NO: 4,the heavy chain CDR2 of SEQ ID NO: 67, the heavy chain CDR3 of SEQ IDNO: 6, and a light chain variable region comprising the light chain CDR1of SEQ ID NO: 68, the light chain CDR2 of SEQ ID NO: 9, and the lightchain CDR3 of SEQ ID NO: 10.

In one embodiment the CD3 binding Fab molecule comprises a heavy chainvariable region sequence that is at least about 95%, 96%, 97%, 98%, 99%or 100% identical to SEQ ID NO: 3 and a light chain variable regionsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to SEQ ID NO: 7.

In one embodiment the CD3 binding Fab molecule comprises a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO: 3 and alight chain variable region comprising the amino acid sequence of SEQ IDNO: 7.

In one embodiment the CD3 binding Fab molecule comprises the heavy chainvariable region sequence of SEQ ID NO: 3 and the light chain variableregion sequence of SEQ ID NO: 7.

Target Cell Antigen Binding Fab Molecule

The T cell activating bispecific antigen binding molecule of theinvention comprises at least one Fab molecule which specifically bindsto a target cell antigen (also referred to herein as “target cellantigen binding Fab molecule”). In certain embodiments, the T cellactivating bispecific antigen binding molecule comprises two Fabmolecules which specifically bind to a target cell antigen. In aparticular such embodiment, each of these Fab molecules specificallybinds to the same antigenic determinant. In an even more particularembodiment, all of these Fab molecules are identical, i.e. they comprisethe same amino acid sequences including the same amino acidsubstitutions in the CH1 and CL domain as described herein (if any). Inone embodiment, the T cell activating bispecific antigen bindingmolecule comprises an immunoglobulin molecule which specifically bindsto a target cell antigen. In one embodiment the T cell activatingbispecific antigen binding molecule comprises not more than two Fabmolecules which specifically bind to a target cell antigen.

In particular embodiments, the Fab molecule(s) which specficially bindto a target cell antigen is/are a conventional Fab molecule. In suchembodiments, the Fab molecule(s) which specifically binds an activatingT cell antigen is a crossover Fab molecule as described herein, i.e. aFab molecule wherein the variable domains VH and VL of the Fab heavy andlight chains are exchanged/replaced by each other.

In alternative embodiments, the Fab molecule(s) which specficially bindto a target cell antigen is/are a crossover Fab molecule as describedherein, i.e. a Fab molecule wherein the variable domains VH and VL ofthe Fab heavy and light chains are exchanged/replaced by each other. Insuch embodiments, the Fab molecule(s) which specifically binds anactivating T cell antigen is a conventional Fab molecule.

The target cell antigen binding Fab molecule binds to a specificantigenic determinant and is able to direct the T cell activatingbispecific antigen binding molecule to a target site, for example to aspecific type of tumor cell that bears the antigenic determinant.

In certain embodiments the target cell antigen binding Fab moleculespecifically binds to a cell surface antigen.

In certain embodiments the target cell antigen binding Fab molecule isdirected to an antigen associated with a pathological condition, such asan antigen presented on a tumor cell or on a virus-infected cell.Suitable target cell antigens are cell surface antigens, for example,but not limited to, cell surface receptors. In particular embodimentsthe target cell antigen is a human antigen. Exemplary target cellantigens include CD20, Her2, Her3, MCSP (melanoma-associated chondroitinsulfate proteoglycan, also known as chondroitin sulfate proteoglycan 4),or BCMA (human B cell maturation target, also known as Tumor NecrosisFactor Receptor Superfamily Member 17 (UniProt Q02223)).

In particular embodiments, the target cell antigen is CD20, particularlyhuman CD20. In one embodiment, the target cell antigen is CD20 and theFab molecule which specifically binds to said target cell antigencomprises a heavy chain variable region comprising the heavy chaincomplementarity determining region (CDR) 1 of SEQ ID NO: 46, the heavychain CDR 2 of SEQ ID NO: 47, and the heavy chain CDR 3 of SEQ ID NO:48, and a light chain variable region comprising the light chain CDR 1of SEQ ID NO: 49, the light chain CDR 2 of SEQ ID NO: 50 and the lightchain CDR 3 of SEQ ID NO: 51. In a further embodiment, the target cellantigen is CD20 and the Fab molecule which specifically binds to saidtarget cell antigen comprises a heavy chain variable region that is atleast 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO:30, and a light chain variable region that is at least 95%, 96%, 97%,98%, or 99% identical to the sequence of SEQ ID NO: 31, In still afurther embodiment, the target cell antigen is CD20 and the Fab moleculewhich specifically binds to said target cell antigen comprises the heavychain variable region sequence of SEQ ID NO: 30, and the light chainvariable region sequence of SEQ ID NO: 31. In a particular embodiment,the T cell activating bispecific antigen binding molecule comprises apolypeptide that is at least 95%, 96%, 97%, 98%, or 99% identical to thesequence of SEQ ID NO: 18, a polypeptide that is at least 95%, 96%, 97%,98%, or 99% identical to the sequence of SEQ ID NO: 19, a polypeptidethat is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence ofSEQ ID NO: 20, and a polypeptide that is at least 95%, 96%, 97%, 98%, or99% identical to the sequence of SEQ ID NO: 21. In a further particularembodiment, the T cell activating bispecific antigen binding moleculecomprises a polypeptide sequence of SEQ ID NO: 18, a polypeptidesequence of SEQ ID NO: 19, a polypeptide sequence of SEQ ID NO: 20 and apolypeptide sequence of SEQ ID NO: 21. In another embodiment, the T cellactivating bispecific antigen binding molecule comprises a polypeptidethat is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence ofSEQ ID NO: 32, a polypeptide that is at least 95%, 96%, 97%, 98%, or 99%identical to the sequence of SEQ ID NO: 19, a polypeptide that is atleast 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO:20, and a polypeptide that is at least 95%, 96%, 97%, 98%, or 99%identical to the sequence of SEQ ID NO: 21. In a further embodiment, thethe T cell activating bispecific antigen binding molecule comprises apolypeptide sequence of SEQ ID NO: 32, a polypeptide sequence of SEQ IDNO: 19, a polypeptide sequence of SEQ ID NO: 20 and a polypeptidesequence of SEQ ID NO: 21. In still another embodiment, the T cellactivating bispecific antigen binding molecule comprises a polypeptidethat is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence ofSEQ ID NO: 36, a polypeptide that is at least 95%, 96%, 97%, 98%, or 99%identical to the sequence of SEQ ID NO: 37, a polypeptide that is atleast 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO:38, and a polypeptide that is at least 95%, 96%, 97%, 98%, or 99%identical to the sequence of SEQ ID NO: 39. In a further embodiment, thethe T cell activating bispecific antigen binding molecule comprises apolypeptide sequence of SEQ ID NO: 36, a polypeptide sequence of SEQ IDNO: 37, a polypeptide sequence of SEQ ID NO: 38 and a polypeptidesequence of SEQ ID NO: 39. In a further embodiment, the T cellactivating bispecific antigen binding molecule comprises a polypeptidethat is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence ofSEQ ID NO: 40, a polypeptide that is at least 95%, 96%, 97%, 98%, or 99%identical to the sequence of SEQ ID NO: 41, a polypeptide that is atleast 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO:20, and a polypeptide that is at least 95%, 96%, 97%, 98%, or 99%identical to the sequence of SEQ ID NO: 21. In a further embodiment, thethe T cell activating bispecific antigen binding molecule comprises apolypeptide sequence of SEQ ID NO: 40, a polypeptide sequence of SEQ IDNO: 41, a polypeptide sequence of SEQ ID NO: 20 and a polypeptidesequence of SEQ ID NO: 21.

In other embodiments, the target antigen is Her2, particularly humanHer2. In one embodiment, the target cell antigen is Her2 and the Fabmolecule which specifically binds to said target cell antigen comprisesa heavy chain variable region that is at least 95%, 96%, 97%, 98%, or99% identical to the sequence of SEQ ID NO: 61, and a light chainvariable region that is at least 95%, 96%, 97%, 98%, or 99% identical tothe sequence of SEQ ID NO: 62, In a further embodiment, the target cellantigen is Her2 and the Fab molecule which specifically binds to saidtarget cell antigen comprises the heavy chain variable region sequenceof SEQ ID NO: 61, and the light chain variable region sequence of SEQ IDNO: 62. In one embodiment, the T cell activating bispecific antigenbinding molecule comprises a polypeptide that is at least 95%, 96%, 97%,98%, or 99% identical to the sequence of SEQ ID NO: 21, a polypeptidethat is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence ofSEQ ID NO: 52, a polypeptide that is at least 95%, 96%, 97%, 98%, or 99%identical to the sequence of SEQ ID NO: 53, and a polypeptide that is atleast 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO:54. In a further embodiment, the T cell activating bispecific antigenbinding molecule comprises a polypeptide sequence of SEQ ID NO: 21, apolypeptide sequence of SEQ ID NO: 52, a polypeptide sequence of SEQ IDNO: 53 and a polypeptide sequence of SEQ ID NO: 54.

In other embodiments, the target antigen is Her3, particularly humanHer3. In one embodiment, the target cell antigen is Her3 and the Fabmolecule which specifically binds to said target cell antigen comprisesa heavy chain variable region that is at least 95%, 96%, 97%, 98%, or99% identical to the sequence of SEQ ID NO: 63, and a light chainvariable region that is at least 95%, 96%, 97%, 98%, or 99% identical tothe sequence of SEQ ID NO: 64, In a further embodiment, the target cellantigen is Her3 and the Fab molecule which specifically binds to saidtarget cell antigen comprises the heavy chain variable region sequenceof SEQ ID NO: 63, and the light chain variable region sequence of SEQ IDNO: 64. In one embodiment, the T cell activating bispecific antigenbinding molecule comprises a polypeptide that is at least 95%, 96%, 97%,98%, or 99% identical to the sequence of SEQ ID NO: 21, a polypeptidethat is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence ofSEQ ID NO: 55, a polypeptide that is at least 95%, 96%, 97%, 98%, or 99%identical to the sequence of SEQ ID NO: 56, and a polypeptide that is atleast 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO:57. In a further embodiment, the T cell activating bispecific antigenbinding molecule comprises a polypeptide sequence of SEQ ID NO: 21, apolypeptide sequence of SEQ ID NO: 55, a polypeptide sequence of SEQ IDNO: 56 and a polypeptide sequence of SEQ ID NO: 57.

In other embodiments, the target antigen is melanoma-associatedchondroitin sulfate proteoglycan (MCSP), particularly human MCSP. In oneembodiment, the target cell antigen is MCSP and the Fab molecule whichspecifically binds to said target cell antigen comprises a heavy chainvariable region that is at least 95%, 96%, 97%, 98%, or 99% identical tothe sequence of SEQ ID NO: 65, and a light chain variable region that isat least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ IDNO: 66, In a further embodiment, the target cell antigen is Her2 and theFab molecule which specifically binds to said target cell antigencomprises the heavy chain variable region sequence of SEQ ID NO: 65, andthe light chain variable region sequence of SEQ ID NO: 66.

In some embodiments, the target antigen is BCMA. In other embodiments,the target cell antigen is not BCMA.

Polynucleotides

The invention further provides isolated polynucleotides encoding a Tcell activating bispecific antigen binding molecule as described hereinor a fragment thereof. In some embodiments, said fragment is an antigenbinding fragment.

The polynucleotides encoding T cell activating bispecific antigenbinding molecules of the invention may be expressed as a singlepolynucleotide that encodes the entire T cell activating bispecificantigen binding molecule or as multiple (e.g., two or more)polynucleotides that are co-expressed. Polypeptides encoded bypolynucleotides that are co-expressed may associate through, e.g.,disulfide bonds or other means to form a functional T cell activatingbispecific antigen binding molecule. For example, the light chainportion of a Fab molecule may be encoded by a separate polynucleotidefrom the portion of the T cell activating bispecific antigen bindingmolecule comprising the heavy chain portion of the Fab molecule, an Fcdomain subunit and optionally (part of) another Fab molecule. Whenco-expressed, the heavy chain polypeptides will associate with the lightchain polypeptides to form the Fab molecule. In another example, theportion of the T cell activating bispecific antigen binding moleculecomprising one of the two Fc domain subunits and optionally (part of)one or more Fab molecules could be encoded by a separate polynucleotidefrom the portion of the T cell activating bispecific antigen bindingmolecule comprising the the other of the two Fc domain subunits andoptionally (part of) a Fab molecule. When co-expressed, the Fc domainsubunits will associate to form the Fc domain.

In some embodiments, the isolated polynucleotide encodes the entire Tcell activating bispecific antigen binding molecule according to theinvention as described herein. In other embodiments, the isolatedpolynucleotide encodes a polypeptides comprised in the T cell activatingbispecific antigen binding molecule according to the invention asdescribed herein.

In certain embodiments the polynucleotide or nucleic acid is DNA. Inother embodiments, a polynucleotide of the present invention is RNA, forexample, in the form of messenger RNA (mRNA). RNA of the presentinvention may be single stranded or double stranded.

Recombinant Methods

T cell activating bispecific antigen binding molecules of the inventionmay be obtained, for example, by solid-state peptide synthesis (e.g.Merrifield solid phase synthesis) or recombinant production. Forrecombinant production one or more polynucleotide encoding the T cellactivating bispecific antigen binding molecule (fragment), e.g., asdescribed above, is isolated and inserted into one or more vectors forfurther cloning and/or expression in a host cell. Such polynucleotidemay be readily isolated and sequenced using conventional procedures. Inone embodiment a vector, preferably an expression vector, comprising oneor more of the polynucleotides of the invention is provided. Methodswhich are well known to those skilled in the art can be used toconstruct expression vectors containing the coding sequence of a T cellactivating bispecific antigen binding molecule (fragment) along withappropriate transcriptional/translational control signals. These methodsinclude in vitro recombinant DNA techniques, synthetic techniques and invivo recombination/genetic recombination. See, for example, thetechniques described in Maniatis et al., MOLECULAR CLONING: A LABORATORYMANUAL, Cold Spring Harbor Laboratory, N.Y. (1989); and Ausubel et al.,CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Greene Publishing Associates andWiley Interscience, N.Y (1989). The expression vector can be part of aplasmid, virus, or may be a nucleic acid fragment. The expression vectorincludes an expression cassette into which the polynucleotide encodingthe T cell activating bispecific antigen binding molecule (fragment)(i.e. the coding region) is cloned in operable association with apromoter and/or other transcription or translation control elements. Asused herein, a “coding region” is a portion of nucleic acid whichconsists of codons translated into amino acids. Although a “stop codon”(TAG, TGA, or TAA) is not translated into an amino acid, it may beconsidered to be part of a coding region, if present, but any flankingsequences, for example promoters, ribosome binding sites,transcriptional terminators, introns, 5′ and 3′ untranslated regions,and the like, are not part of a coding region. Two or more codingregions can be present in a single polynucleotide construct, e.g. on asingle vector, or in separate polynucleotide constructs, e.g. onseparate (different) vectors. Furthermore, any vector may contain asingle coding region, or may comprise two or more coding regions, e.g. avector of the present invention may encode one or more polypeptides,which are post- or co-translationally separated into the final proteinsvia proteolytic cleavage. In addition, a vector, polynucleotide, ornucleic acid of the invention may encode heterologous coding regions,either fused or unfused to a polynucleotide encoding the T cellactivating bispecific antigen binding molecule (fragment) of theinvention, or variant or derivative thereof.

Heterologous coding regions include without limitation specializedelements or motifs, such as a secretory signal peptide or a heterologousfunctional domain. An operable association is when a coding region for agene product, e.g. a polypeptide, is associated with one or moreregulatory sequences in such a way as to place expression of the geneproduct under the influence or control of the regulatory sequence(s).Two DNA fragments (such as a polypeptide coding region and a promoterassociated therewith) are “operably associated” if induction of promoterfunction results in the transcription of mRNA encoding the desired geneproduct and if the nature of the linkage between the two DNA fragmentsdoes not interfere with the ability of the expression regulatorysequences to direct the expression of the gene product or interfere withthe ability of the DNA template to be transcribed. Thus, a promoterregion would be operably associated with a nucleic acid encoding apolypeptide if the promoter was capable of effecting transcription ofthat nucleic acid. The promoter may be a cell-specific promoter thatdirects substantial transcription of the DNA only in predeterminedcells. Other transcription control elements, besides a promoter, forexample enhancers, operators, repressors, and transcription terminationsignals, can be operably associated with the polynucleotide to directcell-specific transcription. Suitable promoters and other transcriptioncontrol regions are disclosed herein. A variety of transcription controlregions are known to those skilled in the art. These include, withoutlimitation, transcription control regions, which function in vertebratecells, such as, but not limited to, promoter and enhancer segments fromcytomegaloviruses (e.g. the immediate early promoter, in conjunctionwith intron-A), simian virus 40 (e.g. the early promoter), andretroviruses (such as, e.g. Rous sarcoma virus). Other transcriptioncontrol regions include those derived from vertebrate genes such asactin, heat shock protein, bovine growth hormone and rabbit â-globin, aswell as other sequences capable of controlling gene expression ineukaryotic cells. Additional suitable transcription control regionsinclude tissue-specific promoters and enhancers as well as induciblepromoters (e.g. promoters inducible tetracyclins). Similarly, a varietyof translation control elements are known to those of ordinary skill inthe art. These include, but are not limited to ribosome binding sites,translation initiation and termination codons, and elements derived fromviral systems (particularly an internal ribosome entry site, or IRES,also referred to as a CITE sequence). The expression cassette may alsoinclude other features such as an origin of replication, and/orchromosome integration elements such as retroviral long terminal repeats(LTRs), or adeno-associated viral (AAV) inverted terminal repeats(ITRs).

Polynucleotide and nucleic acid coding regions of the present inventionmay be associated with additional coding regions which encode secretoryor signal peptides, which direct the secretion of a polypeptide encodedby a polynucleotide of the present invention. For example, if secretionof the T cell activating bispecific antigen binding molecule is desired,DNA encoding a signal sequence may be placed upstream of the nucleicacid encoding a T cell activating bispecific antigen binding molecule ofthe invention or a fragment thereof. According to the signal hypothesis,proteins secreted by mammalian cells have a signal peptide or secretoryleader sequence which is cleaved from the mature protein once export ofthe growing protein chain across the rough endoplasmic reticulum hasbeen initiated. Those of ordinary skill in the art are aware thatpolypeptides secreted by vertebrate cells generally have a signalpeptide fused to the N-terminus of the polypeptide, which is cleavedfrom the translated polypeptide to produce a secreted or “mature” formof the polypeptide. In certain embodiments, the native signal peptide,e.g. an immunoglobulin heavy chain or light chain signal peptide isused, or a functional derivative of that sequence that retains theability to direct the secretion of the polypeptide that is operablyassociated with it. Alternatively, a heterologous mammalian signalpeptide, or a functional derivative thereof, may be used. For example,the wild-type leader sequence may be substituted with the leadersequence of human tissue plasminogen activator (TPA) or mouseβ-glucuronidase.

DNA encoding a short protein sequence that could be used to facilitatelater purification (e.g. a histidine tag) or assist in labeling the Tcell activating bispecific antigen binding molecule may be includedwithin or at the ends of the T cell activating bispecific antigenbinding molecule (fragment) encoding polynucleotide.

In a further embodiment, a host cell comprising one or morepolynucleotides of the invention is provided. In certain embodiments ahost cell comprising one or more vectors of the invention is provided.The polynucleotides and vectors may incorporate any of the features,singly or in combination, described herein in relation topolynucleotides and vectors, respectively. In one such embodiment a hostcell comprises (e.g. has been transformed or transfected with) a vectorcomprising a polynucleotide that encodes (part of) a T cell activatingbispecific antigen binding molecule of the invention. As used herein,the term “host cell” refers to any kind of cellular system which can beengineered to generate the T cell activating bispecific antigen bindingmolecules of the invention or fragments thereof. Host cells suitable forreplicating and for supporting expression of T cell activatingbispecific antigen binding molecules are well known in the art. Suchcells may be transfected or transduced as appropriate with theparticular expression vector and large quantities of vector containingcells can be grown for seeding large scale fermenters to obtainsufficient quantities of the T cell activating bispecific antigenbinding molecule for clinical applications. Suitable host cells includeprokaryotic microorganisms, such as E. coli, or various eukaryoticcells, such as Chinese hamster ovary cells (CHO), insect cells, or thelike. For example, polypeptides may be produced in bacteria inparticular when glycosylation is not needed. After expression, thepolypeptide may be isolated from the bacterial cell paste in a solublefraction and can be further purified. In addition to prokaryotes,eukaryotic microbes such as filamentous fungi or yeast are suitablecloning or expression hosts for polypeptide-encoding vectors, includingfungi and yeast strains whose glycosylation pathways have been“humanized”, resulting in the production of a polypeptide with apartially or fully human glycosylation pattern. See Gerngross, NatBiotech 22, 1409-1414 (2004), and Li et al., Nat Biotech 24, 210-215(2006). Suitable host cells for the expression of (glycosylated)polypeptides are also derived from multicellular organisms(invertebrates and vertebrates). Examples of invertebrate cells includeplant and insect cells. Numerous baculoviral strains have beenidentified which may be used in conjunction with insect cells,particularly for transfection of Spodoptera frugiperda cells. Plant cellcultures can also be utilized as hosts. See e.g. U.S. Pat. Nos.5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describingPLANTIBODIES™ technology for producing antibodies in transgenic plants).Vertebrate cells may also be used as hosts. For example, mammalian celllines that are adapted to grow in suspension may be useful. Otherexamples of useful mammalian host cell lines are monkey kidney CV1 linetransformed by SV40 (COS-7); human embryonic kidney line (293 or 293Tcells as described, e.g., in Graham et al., J Gen Virol 36, 59 (1977)),baby hamster kidney cells (BHK), mouse sertoli cells (TM4 cells asdescribed, e.g., in Mather, Biol Reprod 23, 243-251 (1980)), monkeykidney cells (CV1), African green monkey kidney cells (VERO-76), humancervical carcinoma cells (HELA), canine kidney cells (MDCK), buffalo ratliver cells (BRL 3A), human lung cells (W138), human liver cells (HepG2), mouse mammary tumor cells (MMT 060562), TRI cells (as described,e.g., in Mather et al., Annals N.Y. Acad Sci 383, 44-68 (1982)), MRC 5cells, and FS4 cells. Other useful mammalian host cell lines includeChinese hamster ovary (CHO) cells, including dhfr-CHO cells (Urlaub etal., Proc Natl Acad Sci USA 77, 4216 (1980)); and myeloma cell linessuch as YO, NS0, P3X63 and Sp2/0. For a review of certain mammalian hostcell lines suitable for protein production, see, e.g., Yazaki and Wu,Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press,Totowa, N.J.), pp. 255-268 (2003). Host cells include cultured cells,e.g., mammalian cultured cells, yeast cells, insect cells, bacterialcells and plant cells, to name only a few, but also cells comprisedwithin a transgenic animal, transgenic plant or cultured plant or animaltissue. In one embodiment, the host cell is a eukaryotic cell,preferably a mammalian cell, such as a Chinese Hamster Ovary (CHO) cell,a human embryonic kidney (HEK) cell or a lymphoid cell (e.g., YO, NS0,Sp20 cell).

Standard technologies are known in the art to express foreign genes inthese systems. Cells expressing a polypeptide comprising either theheavy or the light chain of an antigen binding domain such as anantibody, may be engineered so as to also express the other of theantibody chains such that the expressed product is an antibody that hasboth a heavy and a light chain.

In one embodiment, a method of producing a T cell activating bispecificantigen binding molecule according to the invention is provided, whereinthe method comprises culturing a host cell comprising a polynucleotideencoding the T cell activating bispecific antigen binding molecule, asprovided herein, under conditions suitable for expression of the T cellactivating bispecific antigen binding molecule, and recovering the Tcell activating bispecific antigen binding molecule from the host cell(or host cell culture medium).

The components of the T cell activating bispecific antigen bindingmolecule are genetically fused to each other. T cell activatingbispecific antigen binding molecule can be designed such that itscomponents are fused directly to each other or indirectly through alinker sequence. The composition and length of the linker may bedetermined in accordance with methods well known in the art and may betested for efficacy. Examples of linker sequences between differentcomponents of T cell activating bispecific antigen binding molecules arefound in the sequences provided herein. Additional sequences may also beincluded to incorporate a cleavage site to separate the individualcomponents of the fusion if desired, for example an endopeptidaserecognition sequence.

In certain embodiments the one or more antigen binding moieties of the Tcell activating bispecific antigen binding molecules comprise at leastan antibody variable region capable of binding an antigenic determinant.Variable regions can form part of and be derived from naturally ornon-naturally occurring antibodies and fragments thereof. Methods toproduce polyclonal antibodies and monoclonal antibodies are well knownin the art (see e.g. Harlow and Lane, “Antibodies, a laboratory manual”,Cold Spring Harbor Laboratory, 1988). Non-naturally occurring antibodiescan be constructed using solid phase-peptide synthesis, can be producedrecombinantly (e.g. as described in U.S. Pat. No. 4,186,567) or can beobtained, for example, by screening combinatorial libraries comprisingvariable heavy chains and variable light chains (see e.g. U.S. Pat. No.5,969,108 to McCafferty).

Any animal species of antibody, antibody fragment, antigen bindingdomain or variable region can be used in the T cell activatingbispecific antigen binding molecules of the invention. Non-limitingantibodies, antibody fragments, antigen binding domains or variableregions useful in the present invention can be of murine, primate, orhuman origin. If the T cell activating bispecific antigen bindingmolecule is intended for human use, a chimeric form of antibody may beused wherein the constant regions of the antibody are from a human. Ahumanized or fully human form of the antibody can also be prepared inaccordance with methods well known in the art (see e. g. U.S. Pat. No.5,565,332 to Winter). Humanization may be achieved by various methodsincluding, but not limited to (a) grafting the non-human (e.g., donorantibody) CDRs onto human (e.g. recipient antibody) framework andconstant regions with or without retention of critical frameworkresidues (e.g. those that are important for retaining good antigenbinding affinity or antibody functions), (b) grafting only the non-humanspecificity-determining regions (SDRs or a-CDRs; the residues criticalfor the antibody-antigen interaction) onto human framework and constantregions, or (c) transplanting the entire non-human variable domains, but“cloaking” them with a human-like section by replacement of surfaceresidues. Humanized antibodies and methods of making them are reviewed,e.g., in Almagro and Fransson, Front Biosci 13, 1619-1633 (2008), andare further described, e.g., in Riechmann et al., Nature 332, 323-329(1988); Queen et al., Proc Natl Acad Sci USA 86, 10029-10033 (1989);U.S. Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Jones etal., Nature 321, 522-525 (1986); Morrison et al., Proc Natl Acad Sci 81,6851-6855 (1984); Morrison and Oi, Adv Immunol 44, 65-92 (1988);Verhoeyen et al., Science 239, 1534-1536 (1988); Padlan, Molec Immun31(3), 169-217 (1994); Kashmiri et al., Methods 36, 25-34 (2005)(describing SDR (a-CDR) grafting); Padlan, Mol Immunol 28, 489-498(1991) (describing “resurfacing”); Dall'Acqua et al., Methods 36, 43-60(2005) (describing “FR shuffling”); and Osbourn et al., Methods 36,61-68 (2005) and Klimka et al., Br J Cancer 83, 252-260 (2000)(describing the “guided selection” approach to FR shuffling). Humanantibodies and human variable regions can be produced using varioustechniques known in the art. Human antibodies are described generally invan Dijk and van de Winkel, Curr Opin Pharmacol 5, 368-74 (2001) andLonberg, Curr Opin Immunol 20, 450-459 (2008). Human variable regionscan form part of and be derived from human monoclonal antibodies made bythe hybridoma method (see e.g. Monoclonal Antibody Production Techniquesand Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).Human antibodies and human variable regions may also be prepared byadministering an immunogen to a transgenic animal that has been modifiedto produce intact human antibodies or intact antibodies with humanvariable regions in response to antigenic challenge (see e.g. Lonberg,Nat Biotech 23, 1117-1125 (2005). Human antibodies and human variableregions may also be generated by isolating Fv clone variable regionsequences selected from human-derived phage display libraries (see e.g.,Hoogenboom et al. in Methods in Molecular Biology 178, 1-37 (O'Brien etal., ed., Human Press, Totowa, N.J., 2001); and McCafferty et al.,Nature 348, 552-554; Clackson et al., Nature 352, 624-628 (1991)). Phagetypically display antibody fragments, either as single-chain Fv (scFv)fragments or as Fab fragments.

In certain embodiments, the antigen binding moieties useful in thepresent invention are engineered to have enhanced binding affinityaccording to, for example, the methods disclosed in U.S. Pat. Appl.Publ. No. 2004/0132066, the entire contents of which are herebyincorporated by reference. The ability of the T cell activatingbispecific antigen binding molecule of the invention to bind to aspecific antigenic determinant can be measured either through anenzyme-linked immunosorbent assay (ELISA) or other techniques familiarto one of skill in the art, e.g. surface plasmon resonance technique(analyzed on a BIACORE T100 system) (Liljeblad, et al., Glyco J 17,323-329 (2000)), and traditional binding assays (Heeley, Endocr Res 28,217-229 (2002)). Competition assays may be used to identify an antibody,antibody fragment, antigen binding domain or variable domain thatcompetes with a reference antibody for binding to a particular antigen,e.g. an antibody that competes with the V9 antibody for binding to CD3.In certain embodiments, such a competing antibody binds to the sameepitope (e.g. a linear or a conformational epitope) that is bound by thereference antibody. Detailed exemplary methods for mapping an epitope towhich an antibody binds are provided in Morris (1996) “Epitope MappingProtocols,” in Methods in Molecular Biology vol. 66 (Humana Press,Totowa, N.J.). In an exemplary competition assay, immobilized antigen(e.g. CD3) is incubated in a solution comprising a first labeledantibody that binds to the antigen (e.g. V9 antibody, described in U.S.Pat. No. 6,054,297) and a second unlabeled antibody that is being testedfor its ability to compete with the first antibody for binding to theantigen. The second antibody may be present in a hybridoma supernatant.As a control, immobilized antigen is incubated in a solution comprisingthe first labeled antibody but not the second unlabeled antibody. Afterincubation under conditions permissive for binding of the first antibodyto the antigen, excess unbound antibody is removed, and the amount oflabel associated with immobilized antigen is measured. If the amount oflabel associated with immobilized antigen is substantially reduced inthe test sample relative to the control sample, then that indicates thatthe second antibody is competing with the first antibody for binding tothe antigen. See Harlow and Lane (1988) Antibodies: A Laboratory Manualch. 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).

T cell activating bispecific antigen binding molecules prepared asdescribed herein may be purified by art-known techniques such as highperformance liquid chromatography, ion exchange chromatography, gelelectrophoresis, affinity chromatography, size exclusion chromatography,and the like. The actual conditions used to purify a particular proteinwill depend, in part, on factors such as net charge, hydrophobicity,hydrophilicity etc., and will be apparent to those having skill in theart. For affinity chromatography purification an antibody, ligand,receptor or antigen can be used to which the T cell activatingbispecific antigen binding molecule binds. For example, for affinitychromatography purification of T cell activating bispecific antigenbinding molecules of the invention, a matrix with protein A or protein Gmay be used. Sequential Protein A or G affinity chromatography and sizeexclusion chromatography can be used to isolate a T cell activatingbispecific antigen binding molecule essentially as described in theExamples. The purity of the T cell activating bispecific antigen bindingmolecule can be determined by any of a variety of well known analyticalmethods including gel electrophoresis, high pressure liquidchromatography, and the like. For example, the heavy chain fusionproteins expressed as described in the Examples were shown to be intactand properly assembled as demonstrated by reducing SDS-PAGE (see e.g.FIGS. 3A-3Q). Three bands were resolved at approximately Mr 25,000, Mr50,000 and Mr 75,000, corresponding to the predicted molecular weightsof the T cell activating bispecific antigen binding molecule lightchain, heavy chain and heavy chain/light chain fusion protein.

Assays

T cell activating bispecific antigen binding molecules provided hereinmay be identified, screened for, or characterized for theirphysical/chemical properties and/or biological activities by variousassays known in the art.

Affinity Assays

The affinity of the T cell activating bispecific antigen bindingmolecule for an Fc receptor or a target antigen can be determined inaccordance with the methods set forth in the Examples by surface plasmonresonance (SPR), using standard instrumentation such as a BIAcoreinstrument (GE Healthcare), and receptors or target proteins such as maybe obtained by recombinant expression. Alternatively, binding of T cellactivating bispecific antigen binding molecules for different receptorsor target antigens may be evaluated using cell lines expressing theparticular receptor or target antigen, for example by flow cytometry(FACS). A specific illustrative and exemplary embodiment for measuringbinding affinity is described in the following and in the Examplesbelow.

According to one embodiment, K_(D) is measured by surface plasmonresonance using a BIACORE® T100 machine (GE Healthcare) at 25° C.

To analyze the interaction between the Fc-portion and Fc receptors,His-tagged recombinant Fc-receptor is captured by an anti-Penta Hisantibody (Qiagen) immobilized on CMS chips and the bispecific constructsare used as analytes. Briefly, carboxymethylated dextran biosensor chips(CMS, GE Healthcare) are activated withN-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) andN-hydroxysuccinimide (NHS) according to the supplier's instructions.Anti Penta-His antibody is diluted with 10 mM sodium acetate, pH 5.0, to40m/ml before injection at a flow rate of 5 μl/min to achieveapproximately 6500 response units (RU) of coupled protein. Following theinjection of the ligand, 1 M ethanolamine is injected to block unreactedgroups. Subsequently the Fc-receptor is captured for 60 s at 4 or 10 nM.For kinetic measurements, four-fold serial dilutions of the bispecificconstruct (range between 500 nM and 4000 nM) are injected in HBS-EP (GEHealthcare, 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% Surfactant P20,pH 7.4) at 25° C. at a flow rate of 30 μl/min for 120 s.

To determine the affinity to the target antigen, bispecific constructsare captured by an anti human Fab specific antibody (GE Healthcare) thatis immobilized on an activated CMS-sensor chip surface as described forthe anti Penta-His antibody. The final amount of coupled protein is isapproximately 12000 RU. The bispecific constructs are captured for 90 sat 300 nM. The target antigens are passed through the flow cells for 180s at a concentration range from 250 to 1000 nM with a flowrate of 30μl/min. The dissociation is monitored for 180 s.

Bulk refractive index differences are corrected for by subtracting theresponse obtained on reference flow cell. The steady state response wasused to derive the dissociation constant K_(D) by non-linear curvefitting of the Langmuir binding isotherm. Association rates (k_(on)) anddissociation rates (k_(off)) are calculated using a simple one-to-oneLangmuir binding model (BIACORE® T100 Evaluation Software version 1.1.1)by simultaneously fitting the association and dissociation sensorgrams.The equilibrium dissociation constant (K_(D)) is calculated as the ratiok_(off)/k_(on). See, e.g., Chen et al., J Mol Biol 293, 865-881 (1999).

Activity Assays

Biological activity of the T cell activating bispecific antigen bindingmolecules of the invention can be measured by various assays asdescribed in the Examples. Biological activities may for example includethe induction of proliferation of T cells, the induction of signaling inT cells, the induction of expression of activation markers in T cells,the induction of cytokine secretion by T cells, the induction of lysisof target cells such as tumor cells, and the induction of tumorregression and/or the improvement of survival.

Compositions, Formulations, and Routes of Administration

In a further aspect, the invention provides pharmaceutical compositionscomprising any of the T cell activating bispecific antigen bindingmolecules provided herein, e.g., for use in any of the below therapeuticmethods. In one embodiment, a pharmaceutical composition comprises anyof the T cell activating bispecific antigen binding molecules providedherein and a pharmaceutically acceptable carrier. In another embodiment,a pharmaceutical composition comprises any of the T cell activatingbispecific antigen binding molecules provided herein and at least oneadditional therapeutic agent, e.g., as described below.

Further provided is a method of producing a T cell activating bispecificantigen binding molecule of the invention in a form suitable foradministration in vivo, the method comprising (a) obtaining a T cellactivating bispecific antigen binding molecule according to theinvention, and (b) formulating the T cell activating bispecific antigenbinding molecule with at least one pharmaceutically acceptable carrier,whereby a preparation of T cell activating bispecific antigen bindingmolecule is formulated for administration in vivo.

Pharmaceutical compositions of the present invention comprise atherapeutically effective amount of one or more T cell activatingbispecific antigen binding molecule dissolved or dispersed in apharmaceutically acceptable carrier. The phrases “pharmaceutical orpharmacologically acceptable” refers to molecular entities andcompositions that are generally non-toxic to recipients at the dosagesand concentrations employed, i.e. do not produce an adverse, allergic orother untoward reaction when administered to an animal, such as, forexample, a human, as appropriate. The preparation of a pharmaceuticalcomposition that contains at least one T cell activating bispecificantigen binding molecule and optionally an additional active ingredientwill be known to those of skill in the art in light of the presentdisclosure, as exemplified by Remington's Pharmaceutical Sciences, 18thEd. Mack Printing Company, 1990, incorporated herein by reference.Moreover, for animal (e.g., human) administration, it will be understoodthat preparations should meet sterility, pyrogenicity, general safetyand purity standards as required by FDA Office of Biological Standardsor corresponding authorities in other countries. Preferred compositionsare lyophilized formulations or aqueous solutions. As used herein,“pharmaceutically acceptable carrier” includes any and all solvents,buffers, dispersion media, coatings, surfactants, antioxidants,preservatives (e.g. antibacterial agents, antifungal agents), isotonicagents, absorption delaying agents, salts, preservatives, antioxidants,proteins, drugs, drug stabilizers, polymers, gels, binders, excipients,disintegration agents, lubricants, sweetening agents, flavoring agents,dyes, such like materials and combinations thereof, as would be known toone of ordinary skill in the art (see, for example, Remington'sPharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp.1289-1329, incorporated herein by reference). Except insofar as anyconventional carrier is incompatible with the active ingredient, its usein the therapeutic or pharmaceutical compositions is contemplated.

The composition may comprise different types of carriers depending onwhether it is to be administered in solid, liquid or aerosol form, andwhether it need to be sterile for such routes of administration asinjection. T cell activating bispecific antigen binding molecules of thepresent invention (and any additional therapeutic agent) can beadministered intravenously, intradermally, intraarterially,intraperitoneally, intralesionally, intracranially, intraarticularly,intraprostatically, intrasplenically, intrarenally, intrapleurally,intratracheally, intranasally, intravitreally, intravaginally,intrarectally, intratumorally, intramuscularly, intraperitoneally,subcutaneously, subconjunctivally, intravesicularlly, mucosally,intrapericardially, intraumbilically, intraocularally, orally,topically, locally, by inhalation (e.g. aerosol inhalation), injection,infusion, continuous infusion, localized perfusion bathing target cellsdirectly, via a catheter, via a lavage, in cremes, in lipid compositions(e.g. liposomes), or by other method or any combination of the forgoingas would be known to one of ordinary skill in the art (see, for example,Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company,1990, incorporated herein by reference). Parenteral administration, inparticular intravenous injection, is most commonly used foradministering polypeptide molecules such as the T cell activatingbispecific antigen binding molecules of the invention.

Parenteral compositions include those designed for administration byinjection, e.g. subcutaneous, intradermal, intralesional, intravenous,intraarterial intramuscular, intrathecal or intraperitoneal injection.For injection, the T cell activating bispecific antigen bindingmolecules of the invention may be formulated in aqueous solutions,preferably in physiologically compatible buffers such as Hanks'solution, Ringer's solution, or physiological saline buffer. Thesolution may contain formulatory agents such as suspending, stabilizingand/or dispersing agents. Alternatively, the T cell activatingbispecific antigen binding molecules may be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use. Sterile injectable solutions are prepared by incorporatingthe T cell activating bispecific antigen binding molecules of theinvention in the required amount in the appropriate solvent with variousof the other ingredients enumerated below, as required. Sterility may bereadily accomplished, e.g., by filtration through sterile filtrationmembranes. Generally, dispersions are prepared by incorporating thevarious sterilized active ingredients into a sterile vehicle whichcontains the basic dispersion medium and/or the other ingredients. Inthe case of sterile powders for the preparation of sterile injectablesolutions, suspensions or emulsion, the preferred methods of preparationare vacuum-drying or freeze-drying techniques which yield a powder ofthe active ingredient plus any additional desired ingredient from apreviously sterile-filtered liquid medium thereof. The liquid mediumshould be suitably buffered if necessary and the liquid diluent firstrendered isotonic prior to injection with sufficient saline or glucose.The composition must be stable under the conditions of manufacture andstorage, and preserved against the contaminating action ofmicroorganisms, such as bacteria and fungi. It will be appreciated thatendotoxin contamination should be kept minimally at a safe level, forexample, less that 0.5 ng/mg protein. Suitable pharmaceuticallyacceptable carriers include, but are not limited to: buffers such asphosphate, citrate, and other organic acids; antioxidants includingascorbic acid and methionine; preservatives (such asoctadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride; benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionicsurfactants such as polyethylene glycol (PEG). Aqueous injectionsuspensions may contain compounds which increase the viscosity of thesuspension, such as sodium carboxymethyl cellulose, sorbitol, dextran,or the like. Optionally, the suspension may also contain suitablestabilizers or agents which increase the solubility of the compounds toallow for the preparation of highly concentrated solutions.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl cleats or triglycerides, or liposomes.

Active ingredients may be entrapped in microcapsules prepared, forexample, by coacervation techniques or by interfacial polymerization,for example, hydroxymethylcellulose or gelatin-microcapsules andpoly-(methylmethacylate) microcapsules, respectively, in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules) or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences(18th Ed. Mack Printing Company, 1990). Sustained-release preparationsmay be prepared. Suitable examples of sustained-release preparationsinclude semipermeable matrices of solid hydrophobic polymers containingthe polypeptide, which matrices are in the form of shaped articles, e.g.films, or microcapsules. In particular embodiments, prolonged absorptionof an injectable composition can be brought about by the use in thecompositions of agents delaying absorption, such as, for example,aluminum monostearate, gelatin or combinations thereof.

In addition to the compositions described previously, the T cellactivating bispecific antigen binding molecules may also be formulatedas a depot preparation. Such long acting formulations may beadministered by implantation (for example subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, the Tcell activating bispecific antigen binding molecules may be formulatedwith suitable polymeric or hydrophobic materials (for example as anemulsion in an acceptable oil) or ion exchange resins, or as sparinglysoluble derivatives, for example, as a sparingly soluble salt.

Pharmaceutical compositions comprising the T cell activating bispecificantigen binding molecules of the invention may be manufactured by meansof conventional mixing, dissolving, emulsifying, encapsulating,entrapping or lyophilizing processes. Pharmaceutical compositions may beformulated in conventional manner using one or more physiologicallyacceptable carriers, diluents, excipients or auxiliaries whichfacilitate processing of the proteins into preparations that can be usedpharmaceutically. Proper formulation is dependent upon the route ofadministration chosen.

The T cell activating bispecific antigen binding molecules may beformulated into a composition in a free acid or base, neutral or saltform. Pharmaceutically acceptable salts are salts that substantiallyretain the biological activity of the free acid or base. These includethe acid addition salts, e.g., those formed with the free amino groupsof a proteinaceous composition, or which are formed with inorganic acidssuch as for example, hydrochloric or phosphoric acids, or such organicacids as acetic, oxalic, tartaric or mandelic acid. Salts formed withthe free carboxyl groups can also be derived from inorganic bases suchas for example, sodium, potassium, ammonium, calcium or ferrichydroxides; or such organic bases as isopropylamine, trimethylamine,histidine or procaine. Pharmaceutical salts tend to be more soluble inaqueous and other protic solvents than are the corresponding free baseforms.

Therapeutic Methods and Compositions

Any of the T cell activating bispecific antigen binding moleculesprovided herein may be used in therapeutic methods. T cell activatingbispecific antigen binding molecules of the invention can be used asimmunotherapeutic agents, for example in the treatment of cancers.

For use in therapeutic methods, T cell activating bispecific antigenbinding molecules of the invention would be formulated, dosed, andadministered in a fashion consistent with good medical practice. Factorsfor consideration in this context include the particular disorder beingtreated, the particular mammal being treated, the clinical condition ofthe individual patient, the cause of the disorder, the site of deliveryof the agent, the method of administration, the scheduling ofadministration, and other factors known to medical practitioners.

In one aspect, T cell activating bispecific antigen binding molecules ofthe invention for use as a medicament are provided. In further aspects,T cell activating bispecific antigen binding molecules of the inventionfor use in treating a disease are provided. In certain embodiments, Tcell activating bispecific antigen binding molecules of the inventionfor use in a method of treatment are provided. In one embodiment, theinvention provides a T cell activating bispecific antigen bindingmolecule as described herein for use in the treatment of a disease in anindividual in need thereof. In certain embodiments, the inventionprovides a T cell activating bispecific antigen binding molecule for usein a method of treating an individual having a disease comprisingadministering to the individual a therapeutically effective amount ofthe T cell activating bispecific antigen binding molecule. In certainembodiments the disease to be treated is a proliferative disorder. In aparticular embodiment the disease is cancer. In certain embodiments themethod further comprises administering to the individual atherapeutically effective amount of at least one additional therapeuticagent, e.g., an anti-cancer agent if the disease to be treated iscancer. In further embodiments, the invention provides a T cellactivating bispecific antigen binding molecule as described herein foruse in inducing lysis of a target cell, particularly a tumor cell. Incertain embodiments, the invention provides a T cell activatingbispecific antigen binding molecule for use in a method of inducinglysis of a target cell, particularly a tumor cell, in an individualcomprising administering to the individual an effective amount of the Tcell activating bispecific antigen binding molecule to induce lysis of atarget cell. An “individual” according to any of the above embodimentsis a mammal, preferably a human.

In a further aspect, the invention provides for the use of a T cellactivating bispecific antigen binding molecule of the invention in themanufacture or preparation of a medicament. In one embodiment themedicament is for the treatment of a disease in an individual in needthereof. In a further embodiment, the medicament is for use in a methodof treating a disease comprising administering to an individual havingthe disease a therapeutically effective amount of the medicament. Incertain embodiments the disease to be treated is a proliferativedisorder. In a particular embodiment the disease is cancer. In oneembodiment, the method further comprises administering to the individuala therapeutically effective amount of at least one additionaltherapeutic agent, e.g., an anti-cancer agent if the disease to betreated is cancer. In a further embodiment, the medicament is forinducing lysis of a target cell, particularly a tumor cell. In still afurther embodiment, the medicament is for use in a method of inducinglysis of a target cell, particularly a tumor cell, in an individualcomprising administering to the individual an effective amount of themedicament to induce lysis of a target cell. An “individual” accordingto any of the above embodiments may be a mammal, preferably a human.

In a further aspect, the invention provides a method for treating adisease. In one embodiment, the method comprises administering to anindividual having such disease a therapeutically effective amount of a Tcell activating bispecific antigen binding molecule of the invention. Inone embodiment a composition is administered to said individual,comprising the T cell activating bispecific antigen binding molecule ofthe invention in a pharmaceutically acceptable form. In certainembodiments the disease to be treated is a proliferative disorder. In aparticular embodiment the disease is cancer. In certain embodiments themethod further comprises administering to the individual atherapeutically effective amount of at least one additional therapeuticagent, e.g., an anti-cancer agent if the disease to be treated iscancer. An “individual” according to any of the above embodiments may bea mammal, preferably a human.

In a further aspect, the invention provides a method for inducing lysisof a target cell, particularly a tumor cell. In one embodiment themethod comprises contacting a target cell with a T cell activatingbispecific antigen binding molecule of the invention in the presence ofa T cell, particularly a cytotoxic T cell. In a further aspect, a methodfor inducing lysis of a target cell, particularly a tumor cell, in anindividual is provided. In one such embodiment, the method comprisesadministering to the individual an effective amount of a T cellactivating bispecific antigen binding molecule to induce lysis of atarget cell. In one embodiment, an “individual” is a human.

In certain embodiments the disease to be treated is a proliferativedisorder, particularly cancer. Non-limiting examples of cancers includebladder cancer, brain cancer, head and neck cancer, pancreatic cancer,lung cancer, breast cancer, ovarian cancer, uterine cancer, cervicalcancer, endometrial cancer, esophageal cancer, colon cancer, colorectalcancer, rectal cancer, gastric cancer, prostate cancer, blood cancer,skin cancer, squamous cell carcinoma, bone cancer, and kidney cancer.Other cell proliferation disorders that can be treated using a T cellactivating bispecific antigen binding molecule of the present inventioninclude, but are not limited to neoplasms located in the: abdomen, bone,breast, digestive system, liver, pancreas, peritoneum, endocrine glands(adrenal, parathyroid, pituitary, testicles, ovary, thymus, thyroid),eye, head and neck, nervous system (central and peripheral), lymphaticsystem, pelvic, skin, soft tissue, spleen, thoracic region, andurogenital system. Also included are pre-cancerous conditions or lesionsand cancer metastases. In certain embodiments the cancer is chosen fromthe group consisting of renal cell cancer, skin cancer, lung cancer,colorectal cancer, breast cancer, brain cancer, head and neck cancer. Askilled artisan readily recognizes that in many cases the T cellactivating bispecific antigen binding molecule may not provide a curebut may only provide partial benefit. In some embodiments, aphysiological change having some benefit is also consideredtherapeutically beneficial. Thus, in some embodiments, an amount of Tcell activating bispecific antigen binding molecule that provides aphysiological change is considered an “effective amount” or a“therapeutically effective amount”. The subject, patient, or individualin need of treatment is typically a mammal, more specifically a human.

In some embodiments, an effective amount of a T cell activatingbispecific antigen binding molecule of the invention is administered toa cell. In other embodiments, a therapeutically effective amount of a Tcell activating bispecific antigen binding molecule of the invention isadministered to an individual for the treatment of disease.

For the prevention or treatment of disease, the appropriate dosage of aT cell activating bispecific antigen binding molecule of the invention(when used alone or in combination with one or more other additionaltherapeutic agents) will depend on the type of disease to be treated,the route of administration, the body weight of the patient, the type ofT cell activating bispecific antigen binding molecule, the severity andcourse of the disease, whether the T cell activating bispecific antigenbinding molecule is administered for preventive or therapeutic purposes,previous or concurrent therapeutic interventions, the patient's clinicalhistory and response to the T cell activating bispecific antigen bindingmolecule, and the discretion of the attending physician. Thepractitioner responsible for administration will, in any event,determine the concentration of active ingredient(s) in a composition andappropriate dose(s) for the individual subject. Various dosing schedulesincluding but not limited to single or multiple administrations overvarious time-points, bolus administration, and pulse infusion arecontemplated herein.

The T cell activating bispecific antigen binding molecule is suitablyadministered to the patient at one time or over a series of treatments.Depending on the type and severity of the disease, about 1 μg/kg to 15mg/kg (e.g. 0.1 mg/kg-10 mg/kg) of T cell activating bispecific antigenbinding molecule can be an initial candidate dosage for administrationto the patient, whether, for example, by one or more separateadministrations, or by continuous infusion. One typical daily dosagemight range from about 1 μg/kg to 100 mg/kg or more, depending on thefactors mentioned above. For repeated administrations over several daysor longer, depending on the condition, the treatment would generally besustained until a desired suppression of disease symptoms occurs. Oneexemplary dosage of the T cell activating bispecific antigen bindingmolecule would be in the range from about 0.005 mg/kg to about 10 mg/kg.In other non-limiting examples, a dose may also comprise from about 1microgram/kg body weight, about 5 microgram/kg body weight, about 10microgram/kg body weight, about 50 microgram/kg body weight, about 100microgram/kg body weight, about 200 microgram/kg body weight, about 350microgram/kg body weight, about 500 microgram/kg body weight, about 1milligram/kg body weight, about 5 milligram/kg body weight, about 10milligram/kg body weight, about 50 milligram/kg body weight, about 100milligram/kg body weight, about 200 milligram/kg body weight, about 350milligram/kg body weight, about 500 milligram/kg body weight, to about1000 mg/kg body weight or more per administration, and any rangederivable therein. In non-limiting examples of a derivable range fromthe numbers listed herein, a range of about 5 mg/kg body weight to about100 mg/kg body weight, about 5 microgram/kg body weight to about 500milligram/kg body weight, etc., can be administered, based on thenumbers described above. Thus, one or more doses of about 0.5 mg/kg, 2.0mg/kg, 5.0 mg/kg or 10 mg/kg (or any combination thereof) may beadministered to the patient. Such doses may be administeredintermittently, e.g. every week or every three weeks (e.g. such that thepatient receives from about two to about twenty, or e.g. about six dosesof the T cell activating bispecific antigen binding molecule). Aninitial higher loading dose, followed by one or more lower doses may beadministered. However, other dosage regimens may be useful. The progressof this therapy is easily monitored by conventional techniques andassays.

The T cell activating bispecific antigen binding molecules of theinvention will generally be used in an amount effective to achieve theintended purpose. For use to treat or prevent a disease condition, the Tcell activating bispecific antigen binding molecules of the invention,or pharmaceutical compositions thereof, are administered or applied in atherapeutically effective amount. Determination of a therapeuticallyeffective amount is well within the capabilities of those skilled in theart, especially in light of the detailed disclosure provided herein.

For systemic administration, a therapeutically effective dose can beestimated initially from in vitro assays, such as cell culture assays. Adose can then be formulated in animal models to achieve a circulatingconcentration range that includes the IC₅₀ as determined in cellculture. Such information can be used to more accurately determineuseful doses in humans.

Initial dosages can also be estimated from in vivo data, e.g., animalmodels, using techniques that are well known in the art. One havingordinary skill in the art could readily optimize administration tohumans based on animal data.

Dosage amount and interval may be adjusted individually to provideplasma levels of the T cell activating bispecific antigen bindingmolecules which are sufficient to maintain therapeutic effect. Usualpatient dosages for administration by injection range from about 0.1 to50 mg/kg/day, typically from about 0.5 to 1 mg/kg/day. Therapeuticallyeffective plasma levels may be achieved by administering multiple doseseach day. Levels in plasma may be measured, for example, by HPLC.

In cases of local administration or selective uptake, the effectivelocal concentration of the T cell activating bispecific antigen bindingmolecules may not be related to plasma concentration. One having skillin the art will be able to optimize therapeutically effective localdosages without undue experimentation.

A therapeutically effective dose of the T cell activating bispecificantigen binding molecules described herein will generally providetherapeutic benefit without causing substantial toxicity. Toxicity andtherapeutic efficacy of a T cell activating bispecific antigen bindingmolecule can be determined by standard pharmaceutical procedures in cellculture or experimental animals. Cell culture assays and animal studiescan be used to determine the LD₅₀ (the dose lethal to 50% of apopulation) and the ED₅₀ (the dose therapeutically effective in 50% of apopulation). The dose ratio between toxic and therapeutic effects is thetherapeutic index, which can be expressed as the ratio LD₅₀/ED₅₀. T cellactivating bispecific antigen binding molecules that exhibit largetherapeutic indices are preferred. In one embodiment, the T cellactivating bispecific antigen binding molecule according to the presentinvention exhibits a high therapeutic index. The data obtained from cellculture assays and animal studies can be used in formulating a range ofdosages suitable for use in humans. The dosage lies preferably within arange of circulating concentrations that include the ED₅₀ with little orno toxicity. The dosage may vary within this range depending upon avariety of factors, e.g., the dosage form employed, the route ofadministration utilized, the condition of the subject, and the like. Theexact formulation, route of administration and dosage can be chosen bythe individual physician in view of the patient's condition (see, e.g.,Fingl et al., 1975, in: The Pharmacological Basis of Therapeutics, Ch.1, p. 1, incorporated herein by reference in its entirety).

The attending physician for patients treated with T cell activatingbispecific antigen binding molecules of the invention would know how andwhen to terminate, interrupt, or adjust administration due to toxicity,organ dysfunction, and the like. Conversely, the attending physicianwould also know to adjust treatment to higher levels if the clinicalresponse were not adequate (precluding toxicity). The magnitude of anadministered dose in the management of the disorder of interest willvary with the severity of the condition to be treated, with the route ofadministration, and the like. The severity of the condition may, forexample, be evaluated, in part, by standard prognostic evaluationmethods. Further, the dose and perhaps dose frequency will also varyaccording to the age, body weight, and response of the individualpatient.

Other Agents and Treatments

The T cell activating bispecific antigen binding molecules of theinvention may be administered in combination with one or more otheragents in therapy. For instance, a T cell activating bispecific antigenbinding molecule of the invention may be co-administered with at leastone additional therapeutic agent. The term “therapeutic agent”encompasses any agent administered to treat a symptom or disease in anindividual in need of such treatment. Such additional therapeutic agentmay comprise any active ingredients suitable for the particularindication being treated, preferably those with complementary activitiesthat do not adversely affect each other. In certain embodiments, anadditional therapeutic agent is an immunomodulatory agent, a cytostaticagent, an inhibitor of cell adhesion, a cytotoxic agent, an activator ofcell apoptosis, or an agent that increases the sensitivity of cells toapoptotic inducers. In a particular embodiment, the additionaltherapeutic agent is an anti-cancer agent, for example a microtubuledisruptor, an antimetabolite, a topoisomerase inhibitor, a DNAintercalator, an alkylating agent, a hormonal therapy, a kinaseinhibitor, a receptor antagonist, an activator of tumor cell apoptosis,or an antiangiogenic agent.

Such other agents are suitably present in combination in amounts thatare effective for the purpose intended. The effective amount of suchother agents depends on the amount of T cell activating bispecificantigen binding molecule used, the type of disorder or treatment, andother factors discussed above. The T cell activating bispecific antigenbinding molecules are generally used in the same dosages and withadministration routes as described herein, or about from 1 to 99% of thedosages described herein, or in any dosage and by any route that isempirically/clinically determined to be appropriate.

Such combination therapies noted above encompass combined administration(where two or more therapeutic agents are included in the same orseparate compositions), and separate administration, in which case,administration of the T cell activating bispecific antigen bindingmolecule of the invention can occur prior to, simultaneously, and/orfollowing, administration of the additional therapeutic agent and/oradjuvant. T cell activating bispecific antigen binding molecules of theinvention can also be used in combination with radiation therapy.

Articles of Manufacture

In another aspect of the invention, an article of manufacture containingmaterials useful for the treatment, prevention and/or diagnosis of thedisorders described above is provided. The article of manufacturecomprises a container and a label or package insert on or associatedwith the container. Suitable containers include, for example, bottles,vials, syringes, IV solution bags, etc. The containers may be formedfrom a variety of materials such as glass or plastic. The containerholds a composition which is by itself or combined with anothercomposition effective for treating, preventing and/or diagnosing thecondition and may have a sterile access port (for example the containermay be an intravenous solution bag or a vial having a stopper pierceableby a hypodermic injection needle). At least one active agent in thecomposition is a T cell activating bispecific antigen binding moleculeof the invention. The label or package insert indicates that thecomposition is used for treating the condition of choice. Moreover, thearticle of manufacture may comprise (a) a first container with acomposition contained therein, wherein the composition comprises a Tcell activating bispecific antigen binding molecule of the invention;and (b) a second container with a composition contained therein, whereinthe composition comprises a further cytotoxic or otherwise therapeuticagent. The article of manufacture in this embodiment of the inventionmay further comprise a package insert indicating that the compositionscan be used to treat a particular condition. Alternatively, oradditionally, the article of manufacture may further comprise a second(or third) container comprising a pharmaceutically-acceptable buffer,such as bacteriostatic water for injection (BWFI), phosphate-bufferedsaline, Ringer's solution and dextrose solution. It may further includeother materials desirable from a commercial and user standpoint,including other buffers, diluents, filters, needles, and syringes.

EXAMPLES

The following are examples of methods and compositions of the invention.It is understood that various other embodiments may be practiced, giventhe general description provided above.

General Methods

Recombinant DNA Techniques

Standard methods were used to manipulate DNA as described in Sambrook etal., Molecular cloning: A laboratory manual; Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989. The molecularbiological reagents were used according to the manufacturers'instructions. General information regarding the nucleotide sequences ofhuman immunoglobulins light and heavy chains is given in: Kabat, E. A.et al., (1991) Sequences of Proteins of Immunological Interest, 5^(th)ed., NIH Publication No. 91-3242.

DNA Sequencing

DNA sequences were determined by double strand sequencing.

Gene Synthesis

Desired gene segments where required were either generated by PCR usingappropriate templates or were synthesized by Geneart AG (Regensburg,Germany) from synthetic oligonucleotides and PCR products by automatedgene synthesis. In cases where no exact gene sequence was available,oligonucleotide primers were designed based on sequences from closesthomologues and the genes were isolated by RT-PCR from RNA originatingfrom the appropriate tissue. The gene segments flanked by singularrestriction endonuclease cleavage sites were cloned into standardcloning/sequencing vectors. The plasmid DNA was purified fromtransformed bacteria and concentration determined by UV spectroscopy.The DNA sequence of the subcloned gene fragments was confirmed by DNAsequencing. Gene segments were designed with suitable restriction sitesto allow sub-cloning into the respective expression vectors. Allconstructs were designed with a 5′-end DNA sequence coding for a leaderpeptide which targets proteins for secretion in eukaryotic cells.

Example 1

Preparation of T-Cell Bispecific (TCB) Antibodies with and withoutCharge Modifications (Anti-CD20/Anti-CD3)

The following molecules were prepared in this example, schematicillustrations thereof are shown in FIGS. 2A-2K:

-   -   A. “2+1 IgG CrossFab, inverted” without charge modifications        (CH1/CL exchange in CD3 binder) (FIG. 2A, SEQ ID NOs 14-17)    -   B. “2+1 IgG CrossFab, inverted” with charge modifications (VH/VL        exchange in CD3 binder, charge modification in CD20 binders)        (FIG. 2B, SEQ ID NOs 18-21)    -   C. “2+1 IgG CrossFab” with charge modifications (VH/VL exchange        in CD3 binder, charge modification in CD20 binders) (FIG. 2C,        SEQ ID NOs 32, 19-21)    -   D. “2+1 IgG CrossFab, inverted” without charge modifications        (VH/VL exchange in CD3 binder) (FIG. 2D, SEQ ID NOs 33, 15, 17,        21)    -   E. “2+1 IgG CrossFab, inverted” without charge modifications        (VH-CH1/VL-CL exchange in CD3 binder) (FIG. 2E, SEQ ID NOs 34,        15, 17, 35)    -   F. “2+1 IgG CrossFab, inverted” with charge modifications (VH/VL        exchange in CD20 binders, charge modification in CD3 binder)        (FIG. 2F, SEQ ID NOs 36-39)    -   G. “2+1 IgG CrossFab, inverted” with charge modifications and        DDKK mutation in Fc region (VH/VL exchange in CD3 binder, charge        modification in CD20 binders) (FIG. 2G, SEQ ID NOs 40, 41, 20,        21)    -   H. “1+1 CrossMab” with charge modifications (VH/VL exchange in        CD3 binder, charge modification in CD20 binder) (FIG. 2H, SEQ ID        NOs 42, 43, 20, 21)    -   I. “1+1 CrossMab” with charge modifications (VH/VL exchange in        CD3 binder, charge modification in CD20 binder, different CD20        binder) (FIG. 2I, SEQ ID NOs 43-45, 21)    -   J. “2+1 IgG CrossFab, inverted” with charge modifications 213E,        123R (VH/VL exchange in CD3 binder, charge modification in CD20        binder) (FIG. 2J, SEQ ID NOs 69-71, 21)    -   K. “2+1 IgG CrossFab, inverted” with charge modifications (VH/VL        exchange and charge modification in CD3 binder) (FIG. 2K, SEQ ID        NOs 15, 17, 72, 73).

The variable region of heavy and light chain DNA sequences weresubcloned in frame with either the constant heavy chain or the constantlight chain pre-inserted into the respective recipient mammalianexpression vector. Protein expression is driven by an MPSV promoter anda synthetic polyA signal sequence is present at the 3′ end of the CDS.In addition each vector contains an EBV OriP sequence. The moleculeswere produced by co-transfecting HEK293-EBNA cells growing in suspensionwith the mammalian expression vectors using polyethylenimine (PEI). Thecells were transfected with the corresponding expression vectors in a1:2:1:1 ratio (A: “vector heavy chain (VH-CH1-VH-CL-CH2-CH3)”: “vectorlight chain (VL-CL)”: “vector heavy chain (VH-CH1-CH2-CH3)”: “vectorlight chain (VL-CH1)”; B, D, G, J, K: “vector heavy chain(VH-CH1-VL-CH1-CH2-CH3)”: “vector light chain (VL-CL)”: “vector heavychain (VH-CH1-CH2-CH3)”: “vector light chain (VH-CL)”; C: “vector heavychain (VL-CH1-VH-CH1-CH2-CH3)”: “vector light chain (VL-CL)”: “vectorheavy chain (VH-CH1-CH2-CH3)”: “vector light chain (VH-CL)”; E: “vectorheavy chain (VH-CH1-VL-CL-CH2-CH3)”: “vector light chain (VL-CL)”:“vector heavy chain (VH-CH1-CH2-CH3)”: “vector light chain (VH-CH1)”; F:“vector heavy chain (VL-CH1-VH-CH1-CH2-CH3)”: “vector light chain(VH-CL)”: “vector heavy chain (VL-CH1-CH2-CH3)”: “vector light chain(VH-CH1)”) or a 1:1:1:1 ratio (H, I: “vector heavy chain(VL-CH1-CH2-CH3)”: “vector light chain (VL-CL)”: “vector heavy chain(VH-CH1-CH2-CH3)”: “vector light chain (VH-CL)”).

For transfection, HEK293 EBNA cells were cultivated in suspension serumfree in Excell culture medium containing 6 mM L-glutamine and 250 mg/lG418. For the production in 600 ml tubespin flasks (max. working volume400 ml) 600 million HEK293 EBNA cells were seeded 24 hours beforetransfection. For transfection, cells were centrifuged for 5 min at210×g, and supernatant was replaced by 20 ml pre-warmed CD CHO medium.Expression vectors are mixed in 20 ml CD CHO medium to a final amount of400 μg DNA. After addition of 1080 μl PEI solution (2.7 μg/ml) themixture was vortexed for 15 s and subsequently incubated for 10 min atroom temperature. Afterwards cells were mixed with the DNA/PEI solution,transferred to a 600 ml tubespin flask and incubated for 3 hours at 37°C. in an incubator with a 5% CO₂ atmosphere. After incubation, 360 mlExcell+6 mM L-glutamine +5 g/L Pepsoy+1.0 mM VPA medium was added andcells were cultivated for 24 hours. One day after transfection 7% Feed 1(Lonza) was added. After 7 days cultivation supernatant was collectedfor purification by centrifugation for 20-30 min at 3600×g (Sigma 8Kcentrifuge), the solution was sterile filtered (0.22 mm filter) andsodium azide in a final concentration of 0.01% w/v was added. Thesolution was kept at 4° C.

The concentration of the constructs in the culture medium was determinedby ProteinA-HPLC. The basis of separation was binding of Fc-containingmolecules on ProteinA at pH 8.0 and step elution from pH 2.5. There weretwo mobile phases. These were Tris (10 mM)-glycine (50 mM)-NaCl (100 mM)buffers, identical except that they were adjusted to different pHs (8and 2.5). The column body was an Upchurch 2×20 mm pre-column with aninternal volume of ˜63 μl packed with POROS 20A. 100 μl of each samplewas injected on equilibrated material with a flow rate of 0.5 ml/min.After 0.67 minutes the sample was eluted with a pH step to pH 2.5.Quantitation was done by determination of 280 nm absorbance andcalculation using a standard curve with a concentration range of humanIgG₁ from 16 to 166 mg/l.

The secreted protein was purified from cell culture supernatants byaffinity chromatography using Protein A affinity chromatography,followed by a size exclusion chromatographic step.

For affinity chromatography supernatant was loaded on a HiTrap ProteinAHP column (CV=5 mL, GE Healthcare) equilibrated with 25 ml 20 mM sodiumphosphate, 20 mM sodium citrate, pH 7.5. Unbound protein was removed bywashing with at least 10 column volumes 20 mM sodium phosphate, 20 mMsodium citrate, 0.5 M sodium chloride, pH 7.5, followed by an additionalwash step using 6 column volumes 10 mM sodium phosphate, 20 mM sodiumcitrate, 0.5 M sodium chloride, pH 5.45. The column was washedsubsequently with 20 ml 10 mM MES, 100 mM sodium chloride, pH 5.0, andtarget protein was eluted in 6 column volumes 20 mM sodium citrate, 100mM sodium chloride, 100 mM glycine, pH 3.0. Protein solution wasneutralized by adding 1/10 of 0.5 M sodium phosphate, pH 8.0. Targetprotein was concentrated and filtrated prior to loading on a HiLoadSuperdex 200 column (GE Healthcare) equilibrated with 20 mM histidine,140 mM sodium chloride, 0.01% Tween-20, pH 6.0. Molecule A had to bepurified by an additional preparative size exclusion chromatography(SEC) step to achieve a final monomer content of 100%. Therefore,fractions with high monomer content from the first size exclusion stepwere pooled, concentrated and again loaded on a HiLoad Superdex 200column (GE Healthcare). This additional purification step was notnecessary for the other molecules (depending on the side productprofile, however, pooling of fractions and therefore recovery after thefirst size exclusion chromatography was different for these molecules).

Purity and molecular weight of the molecules was analyzed after thefirst purification step (Protein A affinity chromatography) by SDS-PAGEin the absence of a reducing agent and staining with Coomassie(SimpleBlue™ SafeStain, Invitrogen). The NuPAGE® Pre-Cast gel system(Invitrogen, USA) was used according to the manufacturer's instruction(4-12% Tris-Acetate gels or 4-12% Bis-Tris).

The protein concentration of purified protein samples was determined bymeasuring the optical density (OD) at 280 nm, using the molar extinctioncoefficient calculated on the basis of the amino acid sequence.

Purity and molecular weight of molecules after the final purificationstep were analyzed by CE-SDS analyses in the presence and absence of areducing agent. The Caliper LabChip GXII system (Caliper lifescience)was used according to the manufacturer's instruction. 2 μg sample wasused for analyses. The aggregate content of antibody samples wasanalyzed using a TSKgel G3000 SW XL analytical size-exclusion column(Tosoh) in 25 mM K₂HPO₄, 125 mM NaCl, 200 mM L-argininemonohydrocloride, 0.02% (w/v) NaN₃, pH 6.7 running buffer at 25° C.

All molecules were produced and purified following the same method(except for molecule A having been subjected to an additional SEC step,as indicated above).

Molecule A showed a high aggregate content after the first preparativesize exclusion chromatography. The content of aggregates after thispurification step could not be determined since there was no baselineseparation of high molecular weight impurities and the monomericfraction. To obtain 100% monomeric material an additional preparativesize exclusion chromatography step was necessary.

Molecule B was 100% monomeric after one preparative size exclusionchromatography.

The concentration in the supernatant was higher for molecule A, but thefinal yield was (due to the high aggregate content) 2.3 fold lower thanfor molecule B (Table 2).

The final purity shown by CE-SDS analyses was higher for molecule B thanfor molecule A (Table 3, FIGS. 3A and 3B). FIGS. 3M and 3N showchromatograms of the SEC purification step (preparative SEC) whereinmolecule A has a broad peak as compared to molecule B, indicating thatthe preparation of molecule A loaded on the SEC is not homogenous whilethe preparation of molecule B is largely monomeric.

Molecule C could be produced with high titer but compared to molecule Bthe final recovery was lower due to a high content of side products thatcould not be completely removed by the applied chromatography methods(Table 2; Table 3; FIGS. 3B and 3J, and FIGS. 3C and 3K). As shown inFIGS. 3B and 3K, the SDS-PAGE analysis after the Protein A purificationstep showed no side product for molecule B, while the preparation ofmolecule C contains some side products appearing at an apparentmolecular weight of 100 kDa.

Molecule D differs from molecule B only in the absence of the chargedresidues in the anti-CD20 Fabs. This molecule could also be producedtransiently with high titer but as already described for molecule C thefinal quality shown on analytical SEC (98% monomer for molecule D, vs.100% monomer for molecule B) and the recovery was lower than formolecule B due to a high content of side products (Table 2; Table 3;FIGS. 3B and 3J and FIGS. 3D and 3L). As shown in FIGS. 3J and 3L, theSDS-PAGE analysis after the Protein A purification step showed no sideproduct for molecule B, while the preparation of molecule D containssome side products appearing at an apparent molecular weight of 66 kDaand 40 kDa. FIGS. 3N and 3O show chromatograms of the SEC purificationstep (preparative SEC) wherein molecule D has a broad peak as comparedto molecule B, indicating that the preparation of molecule D loaded onthe SEC is not homogenous while the preparation of molecule B is largelymonomeric.

Also the titer of the production of molecule E was high but the finalproduct contained still low molecular weight impurities as shown byanalytical SEC and capillary electrophoresis (Table 2; Table 3; FIG.3E).

In contrast to molecule B, molecule F has the VH-VL exchange on the Fabof the tumor target binding moiety whereas the charge modifications havebeen introduced in the anti-CD3 Fab. This molecule could be producedwith high titers too, but the final recovery was low due to sideproducts. For the anti-CD20/anti-CD3 TCB the format with chargemodifications in the anti-CD20 Fab is preferable with regard toproduction and purification.

Molecule G is a molecule with charge modifications in the Fc region(“DD”=K392D; K409D in one of the subunits of the Fc domain, “KK”=D356K;D399K in the other of the subunits of the Fc domain (EU numbering),replacing the “knob into hole” mutation. The generation of bispecificmolecules is fostered by the introduction of two aspartic acid residueson one heavy chain and two lysine residues in the second heavy chain(FIG. 2G). This molecule could be produced with high titer but the finalproduct has still some high molecular and low molecular weightimpurities shown by analytical SEC and capillary electrophoresis (Table2; Table 3) whereas the side products could be completely removed forthe same molecule carrying the “knob into hole” mutation (molecule B).

Molecule I, which differs from molecule H in its CD20 binder, showed ahigher aggregate content after the final preparative size exclusionchromatography compared to molecule H. The final purity shown by CE-SDSanalyses was higher for molecule H than for the molecule I (Table 3;FIGS. 3H and 31 ). Also the recovery for molecule H was 40% higher thanfor molecule I (Table 2). This result shows that the quality of themolecule is also dependent on the antibody used in the T cell bispecificformat.

The productions of molecule J and molecule K had a good starting titerwhich led to a good yield. However, the final recovery of around 20% forboth molecules was well below the 48% achieved with molecule B (Table2). Both molecules are similar in final quality with >99% monomercontent (Table 2). The purity in non-reduced CE-SDS is better formolecule J (which lacks the charge modifications at position 124 of theCL domain and position 147 of the CH1 domain) with nearly 99% comparedto molecule K (having charge modifications and a VL-VH exchange in theCD3 binder) with 90% (Table 3, FIGS. 3N and 3O). Molecule J showed someprecipitation during the concentration step after affinitychromatography. Molecule K has charge modifications in the CD3 bindingCrossFab rather than the CD20 binding Fabs. This has an impact on thefinal quality as shown by CE-SDS (Table 3, FIG. 3O). The difference inquality is mostly visible after the first purification step on SDS-Page(FIGS. 3P and 3Q). Molecule K contains more side products at 150 kDa and70 kDa (half molecules and constructs probably missing light chains)than molecule J. Both molecules have the same thermal stability which issimilar to molecule B (Table 4).

For the anti-CD20/anti-CD3 TCB the “inverted” version with chargemodifications on the anti CD20 Fab (molecule B) is the format that couldbe produced with the highest recovery and final quality.

TABLE 2 Summary of production and purification of anti-CD20/anti- CD3TCB molecules with and without charge modifications. Analytical App.purity SEC (HMW/ determined Mole- Titer Recovery Yield Monomer/ by LC-MScule (mg/l) [%] (mg/l) LMW) [%] [%] A 16.7 7.2 1.2 0/100/0 * 85-90 * B5.5 48.2 2.8 0/100/0 93 C 25 12.9 3.24 4/93/3 nd D 55 9.8 5.42 2/98/0 ndE 30.5 3.3 0.99 0/96.3/3.7 nd F 57 11.8 6.43 3.4/96.6/0 nd G 56 21 11.83.75/92.3/3.43 nd H 29 9.2 2.66 2/98/0 nd I 52.5 5.8 3.05 2.7/95.3/2 ndJ 77 18 17.4 0.7/99.3/0 nd K 71.5 21.8 15.5 0/99.7/0.3 nd * finalproduct, after two SEC steps

TABLE 3 CE-SDS analyses (non-reduced) of anti-CD20/anti-CD3 TCBmolecules with and without charge modifications. Mole- Peak Size Puritycule # [kDa] [%] A 1 34.13 0.49 2 55.10 0.58 3 58.89 0.97 4 152.30 1.765 165.95 2.25 6 177.64 7.75 7 186.15 14.06 8 194.17 18.37 9 201.68 53.77B 1 160.09 0.57 2 180.70 1.62 3 194.42 97.81 C 1 131.12 0.82 2 141.453.45 3 182.86 2.39 4 192.1 13.5 5 198.13 79.84 D 1 207.04 100 E 1 176.360.67 2 196.54 14.36 3 209.22 84.97 F 1 30.41 0.55 2 65.04 1.33 3 198.802.05 4 203.10 7.94 5 213.93 88.12 G 1 96.50 1.67 2 208.46 96.77 3 216.111.55 H 1 131.98 1.13 2 140.64 1.96 3 153.02 92.24 4 161.24 4.67 I 155.75 1.88 2 158.62 50.78 3 178.6 46.14 4 218.64 1.2 J 1 186.5 1.4 2198.2 98.6 K 1 164.7 4 2 182.4 6 3 200.1 90

Molecular Weight Confirmation by LC-MS Analyses

Deglycosylation

To confirm homogeneous preparation of the molecules, the final proteinsolution was analyzed by LC-MS analyses. To remove heterogeneityintroduced by carbohydrates, the constructs were treated with PNGaseF.For this purpose, the pH of the protein solution was adjusted to pH 7.0by adding 2 μl 2 M Tris to 20 μg protein with a concentration of 0.5mg/ml. 0.8 μg PNGaseF was added and incubated for 12 h at 37° C.

LC-MS Analysis—on Line Detection

The LC-MS method was performed on an Agilent HPLC 1200 coupled to a TOF6441 mass spectrometer (Agilent). The chromatographic separation wasperformed on a Macherey Nagel Polysterene column; RP1000-8 (8 μmparticle size, 4.6×250 mm; cat. No. 719510). Eluent A was 5%acetonitrile and 0.05% (v/v) formic acid in water, eluent B was 95%acetonitrile, 5% water and 0.05% formic acid. The flow rate was 1ml/min, the separation was performed at 40° C. and with 6 (15 μl)protein sample obtained with the treatment described before.

Time % (min.) B  0.5  15 10  60 12.5 100 14.5 100 14.6  15 16  15 16.1100

During the first four minutes the eluate was directed into the waste toprevent salt contamination of the mass spectrometer. The ESI-source wasrunning with a drying gas flow of 12 l/min, a temperature of 350° C. anda nebulizer pressure of 60 psi. The MS spectra were acquired using afragmentor voltage of 380 V and a mass range 700 to 3200 m/z in positiveion mode. MS data are acquired by the instrument software from 4 to 17minutes.

The preparation of molecule A had about 10-15% molecules with mispairedlight chains and traces of free or linked light chains. The preparationof molecule B had traces of molecules comprising two CD3 light chains.Impurities such as free light chain or linked light chain could not bedetected (Table 2).

Thermal Stability by Static Light Scattering

Thermal stability was monitored by Static Light Scattering (SLS) and bymeasuring the intrinsic protein fluorescence in response to appliedtemperature stress. 30 μg of filtered protein sample with a proteinconcentration of 1 mg/ml was applied in duplicate to Optim 2 (AvactaAnalytical Ltd; GB). The temperature was ramped from 25 to 85° C. at0.1° C./min, with the radius and total scattering intensity beingcollected. For determination of intrinsic protein fluorescence thesample was excited at 295 nm and emission was collected between 266 and473 nm. Thermal stability was determined for all molecules, results areshown in Table 4. The aggregation temperature (T_(Agg)) determined bydynamic light scattering and the melting temperature (T_(M)) measured byprotein fluorescence after applying a temperature gradient wascomparable for all molecules with T_(Agg) ranging from 54-58° C. andT_(M) ranging from 56-60° C. (Table 4).

TABLE 4 Thermal stability of anti-CD20/anti-CD3 TCB molecules with andwithout charge modifications. Mole- T_(Aggregation) T_(M) cule [° C.] [°C.] A 54.4 55.9 B 54.3 56.4 C 56 59 D 56 59 E 56 60 F 58 60 G 57 59 H 5556 I 53 57 J 54 55 K 54 55

Binding to CD3 and CD20 of Anti-CD3/Anti-CD20 TCB Antibodies

The binding to CD3 of anti-CD3/anti-CD20 T cell bispecific (TCB)antibodies with or without charge modifications (molecules “A” and “B”above) was measured using human CD3-expressing Jurkat cells. The bindingto CD20 was determined using human CD20-expressing Z-138 cells.Suspension cells were harvested, washed once with PBS, and viability andcell density determined using Vicell. The suspension cells wereresuspended at 2×10⁶ cells/ml in FACS buffer. 100 μl of the cellsuspension were seeded into a 96 well round bottom plate. Each step wasperformed at 4° C. The plates were centrifuged at 360×g for 5 min andthe supernatant was removed. Antibody dilutions were prepared inPBS/0.1% BSA. 30 μl of the diluted anti-CD3/anti-CD20 TCB antibodies orFACS buffer were added to the wells and the cells were incubated for 30min at 4° C. After the incubation, 120 μl FACS buffer were added perwell, the plate was centrifuged for 5 min at 350×g, and the supernatantwas removed. The washing step was repeated once. 30 μl pre-dilutedsecondary antibody was added per well, as indicated in the plate layout.The plates were incubated for further 30 min at 4° C. After theincubation, 120 μl FACS buffer were added per well, the plates werecentrifuged for 5 min at 350×g, and the supernatant was removed. Thewashing step was repeated once for all plates but the plate with Jurkatcells, which were fixed directly after this one washing step. The cellswere fixed using 100 μl BD Fixation buffer per well (#BD Biosciences,554655) at 4° C. for 20-30 min. Cells were re-suspended in 80 μl/wellFACS buffer for the FACS measurement using a BD FACS CantoII.

The result of this experiment is shown in FIG. 4 .

Tumor Cell Lysis and CD4⁺ and CD8⁺ T Cell Activation Upon TCell-Mediated Killing of CD20-Expressing Tumor Target Cells Induced byAnti-CD3/Anti-CD20 TCB Antibodies

T cell-mediated killing of target cells and activation of T cellsinduced by anti-CD3/anti-CD20 TCB antibodies with or without chargemodifications (molecules “A” and “B” above) was assessed on Z-138 andNalm-6 tumor cells. Human PBMCs were used as effectors and killing aswell as T cell activation detected 22 h after incubation with thebispecific antibody. Briefly, target cells were harvested, washed, andplated at density of 30 000 cells/well using round-bottom 96-wellplates. Peripheral blood mononuclear cells (PBMCs) were prepared byHistopaque density centrifugation of fresh blood from healthy humandonors. Fresh blood was diluted with sterile PBS and layered overHistopaque gradient (Sigma, #H8889). After centrifugation (450×g, 30minutes, room temperature), the plasma above the PBMC-containinginterphase was discarded and PBMCs transferred in a new falcon tubesubsequently filled with 50 ml PBS. The mixture was centrifuged (400×g,10 minutes, room temperature), the supernatant discarded and the PBMCpellet washed twice with sterile PBS (centrifugation steps 350×g, 10minutes). The resulting PBMC population was counted automatically(ViCell) and kept in RPMI1640 medium containing 10% FCS and 1%L-alanyl-L-glutamine (Biochrom, K0302) in cell incubator (37° C., 5%CO₂) until further use (no longer than 24 h). For the killing assay, theantibodies were added at indicated concentrations (range of 1000 pM-0.1pM in triplicates). PBMCs were added to target cells at the final E:Tratio of 6:1. After the incubation, plates were centrifuged at 420×g for4 min and 50 μl/well was transferred into fresh 96-flat bottom platesfor LDH detection. LDH detection was performed using a CytotoxicityDetection Kit (Roche #11644793001) according to the instructions of themanufacturer. The remaining cells were washed with PBS containing 0.1%BSA. Surface staining for CD8 (APCCy7 anti-human CD8, Biolegend#301016), CD4 (FITC anti-human CD4, Biolegend #300506), CD69 (BV421anti-human CD69 Biolegend #310930) and CD25 (PECy7 anti-human CD25Biolegend #302612) was performed according to the suppliers'indications. After 30 min at 4° C. cells were washed twice with 150μl/well PBS containing 0.1% BSA and fixed using 100 in/well 2% PFA. Themeasurement was performed using a BD FACS CantoII.

The result of this experiment is shown in FIGS. 5, 6A-6B, and 7A-7B.Both molecules display comparable activity in terms of tumor cell lysisand T cell activation.

B Cell Depletion and CD4⁺ and CD8+ T Cell Activation Upon TCell-Mediated Killing of Healthy Human B Cells Induced byAnti-CD3/Anti-CD20 TCB Antibodies in Human Whole Blood

Human whole blood from a healthy donor was incubated withanti-CD3/anti-CD20 TCB antibodies with or without charge modifications(molecules “A” and “B” above) at indicated concentrations (range of50000 pM-1 pM in triplicates). After 22 h, the blood was mixed and 35 μlwere collected for staining with 20 μl FACS antibody mix containing CD8(APCCy7 anti-human CD8, Biolegend #301016), CD4 (FITC anti-human CD4,Biolegend #300506), CD69 (BV421 anti-human CD69 Biolegend #310930) andCD25 (PECy7 anti-human CD25, Biolegend #302612), CD22 (APC anti-humanCD22, Biolegend #302510) and CD45 (PerCPCy5.5 anti-human CD45, Biolegend#304028). After 15 minutes incubation at room temperature, the blood wasfixed with FACS Lysing solution (BD, #349202) and analyzed by flowcytometry. B cell depletion was calculated based on the ratio of B cellnumbers and CD4⁺ T cell numbers setting the untreated samples to 0% Bcell depletion. The result of this experiment is shown in FIGS. 8 and9A-9B. Both molecules display comparable activity in terms of B celldepletion in the whole blood and T cell activation.

Binding of Anti-CD3/Anti-CD20 TCB Antibody to Human CD20- andCD3-Expressing Target Cells

The binding of the anti-CD3/anti-CD20 TCB antibody shown as molecule “B”above was tested on human CD20-expressing Diffuse Large-Cell B CellLymphoma (DLBCL) cell line (WSU DLCL2, 0.5-1×10⁶ CD20 binding sites) andCD3-expressing immortalized T lymphocyte line (Jurkat). Briefly, cellswere harvested, counted, checked for viability and resuspended at1.5×10⁶ cells/ml in FACS buffer (PBS 0.1% BSA). 100 μl of cellsuspension (containing 0.15×10⁶ cells) were incubated in round-bottom96-well plate for 30 min at 4° C. with increasing concentrations of theCD20 TCB (50 pM-200 nM), washed twice with cold PBS 0.1% BSA,re-incubated for further 30 min at 4° C. with diluted PE-conjugatedAffiniPure F(ab′)2 Fragment goat anti-human IgG Fcg Fragment Specificsecondary antibody (Jackson Immuno Research Lab PE #109-116-170), washedtwice with cold PBS 0.1% BSA, fixed by addition of 2% PFA and analyzedby FACS using a FACS CantoII (Software FACS Diva) excluding dead cellsfrom analysis by FSC/SSC gating.

Results are shown in FIG. 10A (binding to WSU DLCL2 cells) and FIG. 10B(binding to Jurkat cells). Binding curves and the EC50 values related tobinding were calculated using GraphPadPrism5. EC50 values were 0.98 nM(bivalent binding to CD20-expressing WSU DLCL2 cells) and approximately12.5 nM (monovalent binding to CD3-expressing Jurkat cells).

Binding of Anti-CD3/Anti-CD20 TCB Antibody to Human and CynomolgusMonkey CD20- and CD3-Expressing Target Cells

The crossreactivity of the anti-CD3/anti-CD20 TCB antibody shown asmolecule “B” above was evaluated by assessing binding to human andcynomolgus monkey CD20-expressing B cells and CD3-expressing CD4 and CD8T cells. Briefly, heparinized human and cynomolgus monkey blood fromhealthy donors was used to isolate PBMCs by density centrifugation.Isolated PBMCs were counted, checked for viability and resuspended at4×10⁶ cells/ml in FACS buffer (100 μl PBS 0.1% BSA). 100 μl of cellsuspension (containing 0.4×10⁶ cells) were plated into 96-well_U-bottomplate and centrifuged (420×g, 4 min). After removal of the supernatants,PBMCs were incubated for 30 min at 4° C. with increasing concentrationsof the CD20 TCB-AlexaFlour488 (200 pM-200 nM), washed twice with coldPBS 0.1% BSA, re-incubated for further 30 min at 4° C. withhuman/cynomolgus cross-reactive antibodies: anti-CD19 (in house, clone8B8)-AlexaFluor647, anti-CD4 (BD, #552838, clone L200)-PerCPCy5.5 andanti-CD8 (BD, #555367, clone RPA-T8)-PE. After 30 min, PBMCs were washedtwice with cold PBS 0.1% BSA and treated with FACS Lysing solution (BD,#349202) followed by FACS analysis using a FACS CantoII (Software FACSDiva). Binding curves were obtained using GraphPadPrism5.

Results are shown in FIG. 11A (binding to human and cynomolgus monkey Bcells), FIG. 11B (binding to human and cynomolgus monkey CD4 T cells)and FIG. 11C (binding to human and cynomolgus monkey CD8 T cells). TheEC50 values related to binding to CD20-expressing B cells, calculatedusing GraphPadPrism5, were 4.8 nM (human B cells) and 3.3 nM (cynomolgusB cells).

Tumor Cell Lysis Mediated by Different Anti-CD20/Anti-CD3 TCB AntibodyFormats

Tumor cell lysis mediated by different anti-CD20/anti-CD3 TCB antibodyformats (molecules “B”, “A” “C” and “H” shown above) was assessed onZ138 cells (mantle cell lymphoma, 0.06-0.23×10⁶ CD20 binding sites).Human PBMCs were used as effectors and tumor lysis was detected at 21-24h of incubation with the different bispecific antibody formats. Briefly,target cells were harvested, washed, and plated at density of 50 000cells/well using U-bottom 96-well plates. Peripheral blood mononuclearcells (PBMCs) were prepared by Histopaque density centrifugation ofhealthy human blood. Fresh blood was diluted with sterile PBS andlayered over Histopaque gradient (Sigma, #H8889). After centrifugation(450×g, 30 minutes, room temperature, w/o brake), the plasma above thePBMC-containing interphase was discarded and PBMCs transferred in a newfalcon tube subsequently filled with 50 ml of PBS. The mixture wascentrifuged (350×g, 10 minutes, room temperature), the supernatantdiscarded and the PBMC pellet washed with sterile PBS (300×g, 10minutes). The resulting PBMC population was counted automatically(ViCell) and stored in RPMI1640 medium containing 10% FCS and 1%L-alanyl-L-glutamine (Biochrom, K0302) at 37° C., 5% CO₂ in cellincubator until further use (no longer than 24 h). For the tumor lysisassay, the antibodies were added at the indicated concentrations (rangeof 0.1 pM-1 nM in triplicates). PBMCs were added to target cells atfinal E:T ratio of 6:1. Tumor cell lysis was assessed after 21-24 h ofincubation at 37° C., 5% CO₂ by quantification of LDH released into cellsupernatants by apoptotic/necrotic cells (LDH detection kit, RocheApplied Science, #11 644 793 001). Maximal lysis of the target cells(=100%) was achieved by incubation of target cells with 1% Triton X-100.Minimal lysis (=0%) refers to target cells co-incubated with effectorcells without bispecific construct.

FIGS. 12A and 12B shows that different CD20 TCB antibody formats induceda strong and target-specific lysis of CD20⁺ target cells. Panel A showsthat the “CD20 TCB_2+1_with charges, inverted” (molecule “B” shownabove) displays comparable activity to the “CD20 TCB_2+1_no charges,inverted” (molecule “A” shown above) and that both are more potent thanthe “CD20 TCB_1+1_with charges” format (molecule “H” shown above). PanelB shows that “CD20 TCB_2+1_with charges, inverted” (molecule “B” shownabove) is more potent than “CD20 TCB_2+1_with charges, classical” format(molecule “C” shown above). The EC50 values related to killing assays,calculated using GraphPadPrism5 are given in Table 5.

TABLE 5 EC50 values (pM) of tumor cell lysis mediated by differentanti-CD20/anti-CD3 TCB antibody formats evaluated using CD20-expressingZ138 tumor target cells. Panel CD20 antibody format EC50 [pM] A CD20TCB_2 + 1_with charges, inverted 2.18 (molecule B) A CD20 TCB_2 + 1_nocharges, inverted 0.76 (molecule A) A CD20 TCB_1 + 1_with charges 17.54(molecule H) B CD20 TCB_2 + 1_with charges, inverted 0.96 (molecule B) BCD20 TCB_2 + 1_with charges, classical 43.34 (molecule C)

Tumor Cell Lysis and Subsequent T Cell Activation Mediated by DifferentAnti-CD20/Anti-CD3 TCB Antibody Formats

Tumor cell lysis mediated by different anti-CD20/anti-CD3 TCB antibodyformats (molecules “B” and “H” shown above) was further assessed on Z138cells (mantle cell lymphoma) using human PBMCs derived from threedifferent healthy donors as well as on a broader panel of DLBCL linesincluding OCI Ly-18 (0.06-0.2×10⁶ CD20 binding sites), Ramos(0.1-0.4×10⁶ CD20 binding sites), SU-DHL-5 (0.13-0.21×10⁶ CD20 bindingsites), SU-DHL-8 (CD20 binding sites below detection limit of theassay), Toledo (0.02×10⁶ CD20 binding sites) and U2932 (0.09-0.4×10⁶CD20 binding sites) cell lines. Tumor cell harvest, PBMC isolation, andassay conditions were identical to the ones described in the previousexample. E:T ratio for the assays shown in FIGS. 13A-13C was 6:1, forthe assay shown in FIG. 13D it was 3:1. Tumor cell lysis was assessedafter 21 h of incubation at 37° C., 5% CO₂ by quantification of LDHreleased into cell supernatants by apoptotic/necrotic cells (LDHdetection kit, Roche Applied Science, #11 644 793 001). For theassessment of T cell activation occurring upon tumor cell lysis, PBMCswere transferred to a round-bottom 96-well plate, centrifuged at 400×gfor 5 min and washed twice with PBS containing 0.1% BSA. Surfacestaining for CD8 (APCCy7 anti-human CD8 Biolegend, #301016), CD4 (FITCanti-human CD4, Biolegend #300506) and CD25 (PECy7 anti-human CD25Biolegend #302612) was performed according to the suppliers'indications. Cells were washed twice with 150 μl/well PBS containing0.1% BSA and fixed using 2% PFA or FACS Lysing solution (BD, #349202).Samples were analyzed at BD FACS CantoII.

FIGS. 13A-13D shows that the “CD20 TCB_2+1_with charges, inverted”antibody format (molecule “B” shown above) is more potent than “CD20TCB_1+1” antibody format (molecule “H” shown above) as assessed bydetection of both tumor cell lysis (Panels A, D) and T cell activation(Panel B, C) using PBMCs from different donors. The EC50 values relatedto tumor lysis and T cell activation of Z138 cells are given in Table6a. The EC50 values related to tumor lysis assays of a panel of DLBCLcell lines are given in Table 6b. The EC50 values were calculated usingGraphPadPrism5.

TABLE 6a EC50 values (pM) of tumor cell lysis and T cell activationmediated by anti-CD20/anti-CD3 TCB antibodies using CD20-expressing Z138tumor cells. EC50 [pM] 24 h (average of CD20 antibody format 3 donors)CD20 TCB_2 + 1_with charges, inverted 1.6 (tumor lysis) (molecule B)CD20 TCB_1 + 1 (tumor lysis) (molecule H) 751 CD20 TCB_2 + 1_withcharges, inverted 2.2 (CD8 T cell activation) (molecule B) CD20 TCB_1 +1 (CD8 T cell activation) 174.8 (molecule H) CD20 TCB_2 + 1_withcharges, inverted 1.2 (CD4 T cell activation) (molecule B) CD20 TCB_1 +1 (CD4 T cell activation) 122 (molecule H)

TABLE 6b EC50 values (pM) of tumor lysis of a panel of DLBCL tumor celllines mediated by anti-CD20/anti-CD3 TCB antibodies. CD20 EC50 [pM]TCB_2 + 1_with CD20 24 h of charges, inverted TCB_1 + 1 tumor lysis(molecule B) (molecule H) Ocly-18 0.3 250.4 Ramos n.d. n.d. SU-DHL-5 1.2 69.7 SU-DHL-8 0.5 218.9 Toledo n.d. 120.2 U2932 0.9  72.7

B Cell Depletion in Human Whole Blood Mediated by DifferentAnti-CD20/Anti-CD3 TCB Antibody Formats

Normal B cell depletion mediated by different anti-CD20/anti-CD3 TCBantibody formats (molecules “B” and “H” shown above) and by obinutuzumabwas further assessed using fresh human blood from healthy volunteers.Briefly, fresh blood was collected in heparin-containing syringes. Bloodaliquots (180 μL/well) were placed in 96-deep well plates, supplementedwith TCB or antibody dilutions (10 μL/well+10 μL/well PBS) and incubatedfor 24 h at 37° C. in 5% CO₂ in a humidified cell incubator. Afterincubation, blood was mixed by pipetting up and down before 35 μL bloodaliquots were transferred in 96-well U-bottom plates and incubated withfluorescent anti-CD45 (APC, Biolegend, #304037), anti-CD4 (PerCPCy5.5,BD, #552838), anti-CD8 (APCCy7, Biolegend, #301016), anti-CD19 (PE,Biolegend, #302208), anti-CD25 (PECy7, Biolegend, #302612) and anti-CD69(BV421, Biolegend, #310930) in total 55 μL volume for flow cytometry.After 15 min incubation at room temperature (in the dark) 180 μL/well ofFACS lysis solution (BD Biosciences) was added to deplete erythrocytesand to fix cells prior to flow cytometry.

FIG. 14 shows that the “CD20 TCB_2+1_with charges, inverted” (molecule“B” above) is more potent in depleting normal B cells than bothobinutuzumab (Gazyva) and “CD20 TCB_1+1” with charges (molecule “H”above).

TABLE 7 EC50 values (pM) of B cell depletion in normal human whole bloodmediated by different CD20-targeting antibodies. CD20-targetingantibodies EC50 [pM] 24 h CD20 TCB_2 + 1_with charges, 13.2 inverted(molecule B) Obinutuzumab (Gazyva ®) 79.2 CD20 TCB_1 + 1 (molecule H)3753

Activation of T Cells Assessed by Quantification of the Intensity of CD3Downstream Signaling Using Jurkat-NFAT Reporter Assay

The capacity of different anti-CD20/anti-CD3 TCB antibody formats toinduce T cell cross-linking and subsequently T cell activation wasassessed using co-cultures of CD20-expressing tumor target cells andJurkat-NFAT reporter cells (a CD3-expressing human acute lymphaticleukemia reporter cell line with a NFAT promoter, GloResponse JurkatNFAT-RE-luc2P, Promega #CS176501). Upon simultaneous binding ofanti-CD20/anti-CD3 TCB to CD20 antigen (expressed on tumor cells) andCD3 antigen (expressed on Jurkat-NFAT reporter cells), the NFAT promoteris activated and leads to expression of active firefly luciferase. Theintensity of luminescence signal (obtained upon addition of luciferasesubstrate) is proportional to the intensity of CD3 activation andsignaling. Jurkat-NFAT reporter cells grow in suspension and werecultured in RPMI1640, 2 g/l glucose, 2 g/l NaHCO₃, 10% FCS, 25 mM HEPES,2 mM L-glutamin, 1×NEAA, 1× sodium-pyruvate at 0.1-0.5 mio cells per ml,200 μg per ml hygromycin. For the assay, tumor target cells (Z138) wereharvested and viability determined using ViCell. 50 μl/well of dilutedantibodies or medium (for controls) was added to target cells. 20 000cells/well were plated in a flat-bottom, white-walled 96-well-plate(#655098, Greiner bio-one). Subsequently, Jurkat-NFAT reporter cellswere harvested and viability assessed using ViCell. Cells wereresuspended at 2 mio cells/ml in cell culture medium without hygromycinB and added to tumor cells at 0.1×10⁶ cells/well (50 μl/well) to obtaina final E:T of 5:1 and a final volume of 100 μl per well. Cells wereincubated for 6 h at 37° C. in a humidified incubator. At the end ofincubation time, 100 μl/well of ONE-Glo solution (1:1 ONE-Glo and assaymedium volume per well) were added to wells and incubated for 10 min atroom temperature in the dark. Luminescence was detected using WALLACVictor3 ELISA reader (PerkinElmer2030), 5 sec/well as detection time.FIG. 15 shows that “CD20 TCB_2+1_with charges, inverted” (molecule “B”above) leads to stronger T cell activation and signaling downstream ofCD3 than “CD20 TCB_1+1” (molecule “H” above).

TABLE 8 EC50 values (pM) of CD3 activation detected using Jurkat-NFATreporter cells. CD20 antibody format EC50 [pM] CD20 TCB_2 + 1_withcharges, 28.98 inverted (molecule B) CD20 TCB_1 + 1 (molecule H) 1001

Single Dose PK of Anti-CD20/Anti-CD3 TCB in Healthy NOG Mice

A single dose pharmacokinetic study (SDPK) was performed to evaluateexposure of anti-CD20/anti-CD3 TCB molecule “B” (hereinafter called“CD20 TCB”) during efficacy studies (FIG. 16 ). An i.v. bolusadministration of 0.5 mg/kg was administered to NOG mice and bloodsamples were taken at selected time points for pharmacokineticevaluation. A generic immunoassay was used for measuring totalconcentrations of the CD20 TCB. The calibration range of the standardcurve for the CD20 TCB was 0.78 to 50 ng/ml, where 15 ng/ml is the lowerlimit of quantification (LLOQ).

A biphasic decline was observed with a beta half-life of 10 days(non-compartmental analysis) and clearance of 8 mL/d/kg (2-compartmentalmodel). The half-life and clearance was as expected as compared to anormal untargeted IgG (Table 9).

Phoenix v6.2 from Pharsight Ltd was used for PK analysis, modelling andsimulation.

TABLE 9 Pharmacokinetic parameters of a 0.5 mg/kg i.v. bolusadministration of CD20 TCB in NOG mice. Half-life 10 days Clearance 7.9mL/d/kg Cmax 9.4 ug/mL AUC 1554 h*ug/mL

In Vivo B-Cell Depletion Activity of Anti-CD20/Anti-CD3 TCB

Peripheral B-cell depletion activity of CD20 TCB was tested in fullyhumanized NOD/Shi-scid/IL-2Rγ^(null) (NOG) mice.

Fully humanized NOG mice at 14 weeks of age, bearing physiologicallevels of circulating human B- and T-cells (Hayakawa J. et al. (2009),Stem Cells 27(1), 175-182), were treated either with vehicle (n=7) orwith CD20 TCB (n=6) at the dose of 0.5 mg/kg administered intravenously(i.v.) once per week. As shown on the study design in FIG. 17 , micewere bled for B cell and T-cell analysis one and three days after thefirst therapeutic injection (D1, D3), and three days after the second(D10), at which time point the study was terminated. At the latter timepoint, spleens were also harvested for B-cell and T-cell analysis Micewere screened 4 days before therapeutic injection (D-4) as baselinereference for circulating B- and T-cell counts. FIGS. 18A and 18B showsB- and T-cell counts analysed by ex vivo flow cytometry in blood ofvehicle (left panel) and CD20 TCB (right panel)-treated mice at thedifferent time points. Results demonstrate that circulating B-cells werevery efficiently depleted already one day after CD20 TCB injection, andtheir number remained undetectable for the whole study duration. On thecontrary, circulating T-cell count only transiently dropped at D1 aftertherapeutic injection, returned to baseline levels at D3, and remainedstable for the whole study duration. T-cell activation status was alsoanalysed in blood of treated mice at D3 and D10 after first therapeuticinjection, by means of ex vivo flow cytometry using different T-cellsurface markers and the proliferation marker Ki67 (FIG. 19 ). T-cellsfrom CD20 TCB-treated mice showed an activated phenotype at D3 aftertherapeutic injection (upper panel), with up-regulation of theactivation markers CD25, 4-1BB, PD-1 and granzyme-B (GZB) in both CD4and CD8 T-cell compartments, compared to T-cells from vehicle control.T-cells from treated mice also expressed higher levels of theproliferation marker Ki67. At D10 after first therapeutic injection,most of the T-cell activation markers had returned to baseline levelswith the exception of GZB and PD-1, which were still expressed at higherlevels compared to vehicle control.

FIGS. 20A-20C shows the results of B-cell and T-cell analyses done onspleens of vehicle and CD20 TCB-treated mice at study termination (D10).CD20 TCB treatment mediated a very efficient B cell depletion also inthis secondary lymphoid organ (FIG. 20A), while T-cell counts showedlevels comparable to vehicle control (FIG. 20B). The T cell activationstatus (FIG. 20C) was similar to that observed in blood, with higherexpression of GZB and PD-1 in splenic T cells of treated mice comparedto vehicle control.

Altogether these results demonstrate that CD20 TCB treatment can mediatea very efficient depletion of peripheral B-cells already one day aftertherapy injection, with B-cells remaining undetectable until studytermination (three days after second therapeutic injection). B-cells arealso efficiently depleted in spleen of treated mice. B-cell depletionactivity is paralleled by a transient T-cell activation in blood oftreated animals, which returns to baseline levels three days aftertherapeutic injection, with the exception of GZB and PD-1 activationmarkers, which remain expressed at a higher level compared to untreatedcontrols.

Anti-Tumor Activity of Anti-CD20/Anti-CD3 TCB in WSU-DLCL2 Model

Anti-tumor activity of CD20 TCB was tested in NOG mice bearing the humandiffuse large B cell lymphoma cell line WSU-DLCL2 and transferred withhuman peripheral mononuclear cells (PBMC). Briefly, female NOG mice wereinjected sub-cutaneously (s.c.) with 1.5×10⁶ WSU-DLCL2 cells (originallyobtained from the European Collection of Cell Culture). When averagetumor volume reached 200 mm³, mice received intra-peritoneal injectionof human PBMC (10×10⁶ cells per mouse) as source human T-cells. Two dayslater, mice received CD20 TCB therapy i.v. at a dose of 0.5 mg/kgadministered once a week. As depicted in FIG. 21 , CD20 TCB shows apotent anti-tumor activity, with almost complete tumor regressionobserved at study termination (day 34).

Example 2

Preparation of “2+1 IgG CrossFab, Inverted” T-Cell Bispecific Antibodywith and without Charge Modifications (Anti-BCMA/Anti-CD3)

Schematic illustrations of the molecules prepared in this example areshown in FIG. 22 . The anti-BCMA/anti-CD3 “2+1 IgG CrossFab, inverted”molecule without charge modifications (referred to in this example as“83A10-TCB”) comprises the amino acid sequences of SEQ ID NOs 22-25, theanti-BCMA/anti-CD3 “2+1 IgG CrossFab, inverted” molecule with chargemodifications (referred to in this example as “83A10-TCBcv”) comprisesthe amino acid sequences of SEQ ID NOs 26-29.

For the generation of BCMAxCD3 bispecific antibody vectors, the IgG₁derived bispecific molecules consist at least of two antigen bindingmoieties capable of binding specifically to two distinct antigenicdeterminants CD3 and BCMA. The antigen binding moieties are Fabfragments composed of a heavy and a light chain, each comprising avariable and a constant region. At least one of the Fab fragments was a“Crossfab” fragment, wherein VH and VL were exchanged. The exchange ofVH and VL within the Fab fragment assures that Fab fragments ofdifferent specificity do not have identical domain arrangements. Thebispecific molecule design was monovalent for CD3 and bivalent for BCMAwhere one Fab fragment was fused to the N-terminus of the inner CrossFab(2+1). The bispecific molecule contained an Fc part in order for themolecule to have a longer half-life. The molecules were produced byco-transfecting HEK293 EBNA cells growing in suspension with themammalian expression vectors using polyethylenimine (PEI). Forpreparation of 2+1 CrossFab-IgG constructs, cells were transfected withthe corresponding expression vectors in a 1:2:1:1 ratio (“vectorFc(knob)”: “vector light chain”: “vector light chain CrossFab”: “vectorheavy chain-CrossFab”).

For bispecific antibodies, introduction of a replacement/exchange in onebinding arm “Crossfab” clearly reduces the side-products but thepreparation is not completely free of side-products (described in detailin WO2009/080252 and Schaefer, W. et al, PNAS, 108 (2011) 11187-1191).Thus, to further reduce side-products caused by the mismatch of a lightchain against a first antigen with the wrong heavy chain against thesecond antigen and to improve the yield of the bispecific antibody, anadditional approach is applied to the molecule by introducingsubstitutions of charged amino acids with the opposite charge atspecific amino acid positions in the CH1 and CL domains in the constantdomain CL of the first light chain under a) the amino acid at position124 is substituted independently by lysine (K), arginine (R) orhistidine (H) (numbering according to Kabat) (in one preferredembodiment independently by lysine (K), arginine (R)), and wherein inthe constant domain CH1 of the first heavy chain under a) the amino acidat position 147 or the amino acid at position 213 is substitutedindependently by glutamic acid (E), or aspartic acid (D) (numberingaccording to Kabat); or ii) in the constant domain CL of the secondlight chain under b) the amino acid at position 124 is substitutedindependently by lysine (K), arginine (R) or Histidine (H) (numberingaccording to Kabat) (in one preferred embodiment independently by lysine(K), arginine (R)), and wherein in the constant domain CH1 of the secondheavy chain under b) the amino acid at positions 147 or the amino acidat position 213 is substituted independently by glutamic acid (E), oraspartic acid (D) (numbering according to Kabat).

For the production of the bispecific antibodies, bispecific antibodieswere expressed by transient co-transfection of the respective mammalianexpression vectors in HEK293-EBNA cells, which were cultivated insuspension, using polyethylenimine (PEI). One day prior to transfection,the HEK293-EBNA cells were seeded at 1.5 Mio viable cells/mL in Ex-Cellmedium supplemented with 6 mM of L-Glutamine. For every mL of finalproduction volume, 2.0 Mio viable cells were centrifuged (5 minutes at210×g). The supernatant was aspirated and the cells resuspended in 100μL of CD CHO medium. The DNA for every mL of final production volume wasprepared by mixing 1 μg of DNA (Ratio heavy chain:modified heavychain:light chain:modified light chain=1:1:2:1) in 100 μL of CD CHOmedium. After addition of 0.27 μL of PEI solution (1 mg/mL) the mixturewas vortexed for 15 seconds and left at room temperature for 10 minutes.After 10 minutes, the resuspended cells and DNA/PEI mixture were puttogether and then transferred into an appropriate container which wasplaced in a shaking device (37° C., 5% CO₂). After a 3 hours incubationtime 800 μL of Ex-Cell Medium, supplemented with 6 mM L-Glutamine, 1.25mM valproic acid and 12.5% Pepsoy (50 g/L), was added for every mL offinal Production volume. After 24 hours, 70 μL of Feed (SF40, Lonza) wasadded for every mL of final production volume. After 7 days or when thecell viability was equal or lower than 70%, the cells were separatedfrom the supernatant by centrifugation and sterile filtration. Theantibodies were purified by an affinity step and one or two polishingsteps, being cation exchange chromatography and size exclusionchromatography. When required, an additional polishing step was used.

For the affinity step the supernatant was loaded on a protein A column(HiTrap Protein A FF, 5 mL, GE Healthcare) equilibrated with 6 CV 20 mMsodium phosphate, 20 mM sodium citrate, pH 7.5. After a washing stepwith the same buffer the antibody was eluted from the column by stepelution with 20 mM sodium phosphate, 100 mM sodium chloride, 100 mMGlycine, pH 3.0. The fractions with the desired antibody wereimmediately neutralized by 0.5 M Sodium Phosphate, pH 8.0 (1:10), pooledand concentrated by centrifugation. The concentrate was sterile filteredand processed further by cation exchange chromatography and/or sizeexclusion chromatography.

For the cation exchange chromatography step the concentrated protein wasdiluted 1:10 with the elution buffer used for the affinity step andloaded onto a cation exchange column (Poros 50 HS, Applied Biosystems).After two washing steps with the equilibration buffer and a washingbuffer resp. 20 mM sodium phosphate, 20 mM sodium citrate, 20 mM TRIS,pH 5.0 and 20 mM sodium phosphate, 20 mM sodium citrate, 20 mM TRIS, 100mM sodium chloride pH 5.0 the protein was eluted with a gradient using20 mM sodium phosphate, 20 mM sodium citrate, 20 mM TRIS, 100 mM sodiumchloride pH 8.5. The fractions containing the desired antibody werepooled, concentrated by centrifugation, sterile filtered and processedfurther a size exclusion step.

For the size exclusion step the concentrated protein was injected in aXK16/60 HiLoad Superdex 200 column (GE Healthcare), and 20 mM Histidine,140 mM Sodium Chloride, pH 6.0 with or without Tween20 as formulationbuffer. The fractions containing the monomers were pooled, concentratedby centrifugation and sterile filtered into a sterile vial.

Determination of the antibody concentration was done by measurement ofthe absorbance at 280 nm, using the theoretical value of the absorbanceof a 0.1% solution of the antibody. This value was based on the aminoacid sequence and calculated by GPMAW software (Lighthouse data).

Purity and monomer content of the final protein preparation wasdetermined by CE-SDS (Caliper LabChip GXII system (Caliper LifeSciences)) resp. HPLC (TSKgel G3000 SW XL analytical size exclusioncolumn (Tosoh)) in a 25 mM potassium phosphate, 125 mM Sodium chloride,200 mM L-arginine monohydrochloride, 0.02% (w/v) Sodium azide, pH 6.7buffer.

To verify the molecular weight of the final protein preparations andconfirm the homogeneous preparation of the molecules final proteinsolution, liquid chromatography-mass spectometry (LC-MS) was used. Adeglycosylation step was first performed. To remove heterogeneityintroduced by carbohydrates, the constructs were treated with PNGaseF(ProZyme). Therefore, the pH of the protein solution was adjusted topH7.0 by adding 2 μl 2 M Tris to 20 μg protein with a concentration of0.5 mg/ml. 0.8 μg PNGaseF was added and incubated for 12 h at 37° C. TheLC-MS online detection was then performed. LC-MS method was performed onan Agilent HPLC 1200 coupled to a TOF 6441 mass spectrometer (Agilent).The chromatographic separation was performed on a Macherey NagelPolysterene column; RP1000-8 (8 μm particle size, 4.6×250 mm; cat. No.719510). Eluent A was 5% acetonitrile and 0.05% (v/v) formic acid inwater, eluent B was 95% acetonitrile, 5% water and 0.05% formic acid.The flow rate was 1 ml/min, the separation was performed at 40° C. and 6μg (15 μl) of a protein sample obtained with a treatment as describedbefore.

Time % (min.) B  0.5 15 10 60 12.5 100 14.5 100 14.6 15 16 15 16.1 100

During the first 4 minutes, the eluate was directed into the waste toprotect the mass spectrometer from salt contamination. The ESI-sourcewas running with a drying gas flow of 12 l/min, a temperature of 350° C.and a nebulizer pressure of 60 psi. The MS spectra were acquired using afragmentor voltage of 380 V and a mass range 700 to 3200 m/z in positiveion mode using. MS data were acquired by the instrument software from 4to 17 minutes.

FIGS. 23A-23C depicts the CE-SDS (non-reduced) graphs of the finalprotein preparations after different methods of purification for83A10-TCB and 83A10-TCBcv antibodies. Protein A (PA) affinitychromatography and size exclusion chromatographic (SEC) purificationsteps applied to 83A10-TCB antibody resulted in a purity of <30% and82.8% of monomer content (A). When additional purifications stepsincluding cation exchange chromatography (cIEX) and a final sizeexclusion chromatographic (re-SEC) steps were applied to the finalprotein preparations in (A), the purity was increased to 93.4% but themonomer content remained the same and the yield was significantlyreduced to 0.42 mg/L. However, when specific charge modifications wereapplied to 83A10 anti-BCMA Fab CL-CH1, namely 83A10-TCBcv antibody, asuperior production/purification profile of the TCB molecule, asdemonstrated by a purity of 95.3%, monomer content of 100% and yield ofup to 3.3 mg/L, could already be observed even when PA+cIEX+SECpurification steps were applied (C) in comparison to (B) with aproduction/purification profile showing a 7.9-fold lower yield and 17.2%lower monomer content despite including an additional re-SECpurification step.

A head-to-head production run to compare the production/purificationprofile of 83A10-TCB vs. 83A10-TCBcv antibodies was then conducted tofurther evaluate the advantages of the CL-CH1 charge modificationsapplied to the antibodies. As depicted in FIGS. 24A-24F, properties of83A10-TCB and 83A10-TCBcv antibodies were measured side-by-side andcompared after each purification steps 1) PA affinity chromatographyonly (A, B), 2) PA affinity chromatography then SEC (C, D) and 3) PAaffinity chromatography then SEC then cIEX and re-SEC (E, F). The CE-SDS(non-reduced) graphs of the final protein solutions after the respectivemethods of purification for 83A10-TCB and 83A10-TCBcv antibodies aredemonstrated in FIGS. 24A-24F. As shown in FIGS. 24A and 24B,improvements with applying the charge variants to the TCB antibody werealready observed after purification by PA affinity chromatography only.In this head-to-head study, PA affinity chromatography purification stepapplied to 83A10-TCB antibody resulted in a purity of 61.3%, a yield of26.2 mg/L and 63.7% of monomer content (24A). In comparison, when83A10-TCBcv antibody was purified by PA affinity chromatography all theproperties were improved with a better purity of 81.0%, a better yieldof 51.5 mg/L and 68.2% of monomer content (24B). When an additional SECpurifications step was applied to the final protein preparations as seenin FIGS. 24A and 24B, 83A10-TCB gained a purity of 69.5%, a yield of14.1 mg/L and 74.7% of monomer content as compared to 83A10-TCBcv withimproved purity and monomer content of up to 91.0% and 83.9%respectively, and a yield of 10.3 mg/L. Even though the yield wasslightly less (i.e. 27% less) for 83A10-TCBcv than for 83A10-TCB in thisparticular experiment, the percentage of correct molecule was muchbetter for 83A10-TCBcv than for 83A10-TCB, respectively 90% vs. 40-60%,as measured by LC-MS. In the third head-to-head comparison, 83A10-TCBand 83A10-TCBcv final protein preparations from FIGS. 24C and 24D werepooled with approximately 1 L (equivolume) of respective final proteinpreparations from another purification batch (same production) followingPA affinity chromatography purification step only. The pooled proteinpreparations were then being further purified by cIEX and SECpurification methods. As depicted in FIGS. 24E and 24F, improvement ofthe production/purification profile of the TCB antibody with the chargevariants was consistently observed when compared to TCB antibody withoutcharge variant. After several steps of purification methods (i.e.PA+/−SEC+cIEX+SEC) were used to purify 83A10-TCB antibody, only 43.1%purity was reached and 98.3% of monomer content could be achieved but tothe detriment of the yield which was reduced to 0.43 mg/L. Thepercentage of correct molecule as measured by LC-MS was still poor with60-70%. At the end, the quality of the final protein preparation was notacceptable for in vitro use. In stark contrast, when the same multiplepurification steps with the same chronology were applied to 83A10-TCBcvantibody, 96.2% purity and 98.9% of monomer content were reached as wellas 95% of correct molecule as measured by LC-MS. The yield however wasalso greatly reduced to 0.64 mg/L after cIEX purification step. Theresults show that better purity, higher monomer content, higherpercentage of correct molecule and better yield can be achieved with83A10-TCBcv antibody only after two standard purification steps i.e. PAaffinity chromatography and SEC (FIG. 24D) while such properties couldnot be achieved with 83A10-TCB even when additional purification stepswere applied (FIG. 24E).

Table 10 summarizes the properties of 83A10-TCB as compared to83A10-TCVcv following PA purification step. Table 11 summarizes theproperties of 83A10-TCB as compared to 83A10-TCVcv following PA and SECpurification steps. Table 12 summarizes the properties of 83A10-TCB ascompared to 83A10-TCVcv following PA and SEC plus PA alone then cIEX andre-SEC purification steps. For Tables 10 to 12, the values in boldhighlight the superior property as compared between 83A10-TCB vs.83A10-TCVcv. With one exception which may not be representative, all theproduction/purification parameters and values resulting from the 3head-to-head comparison experiments were superior for 83A10-TCBcv ascompared to 83A10-TCB. The overall results clearly demonstrate thatadvantages in production/purification features could be achieved withapplying CL-CH1 charge modifications to TCB antibodies and that only twopurification steps (i.e PA affinity chromatography and SEC) wererequired to achieve already high quality protein preparations with verygood developability properties.

TABLE 10 Production/purification profile of anti-BCMA/anti- CD3 T cellbispecific antibodies following protein A affinity chromatographypurification step. 83A10-TCB 83A10-TCBcv Purity (%) 61.3 81.0 Yield(mg/L) 26.2 51.5 Amount (mg) 24.3 50.2 Monomer (%) 63.7 68.2 Correctmolecule n.d. n.d by LC-MS (%)

TABLE 11 Production/purification profile of anti-BCMA/anti-CD3 T cellbispecific antibodies following protein A affinity chromatography andsize exclusion chromatography purification steps. 83A10-TCB 83A10-TCBcvPurity (%) 69.5 91.0 Yield (mg/L) 14.1 10.3 Amount (mg) 13.1 10.0Monomer (%) 74.7 83.9 Correct molecule 40-60 90 by LC-MS (%)

TABLE 12 Production/purification profile of anti-BCMA/anti-CD3 T cellbispecific antibodies following 1.a) protein A affinity chromatographyand size exclusion chromatography and 1.b) protein A affinitychromatography only pooled together then 2) cation exchangechromatography and 3) final size exclusion chromatography purificationsteps. 83A10-TCB 83A10-TCBcv Purity (%) 43.1 96.2 Yield (mg/L) 0.43 0.64Amount (mg) 0.73 1.27 Monomer (%) 98.3 98.9 Correct molecule 60-70% >95%by LC-MS (%)

Binding of Anti-BCMA/Anti-CD3 T-Cell Bispecific Antibodies toBCMA-Positive Multiple Myeloma Cell Lines (Flow Cytometry)

Anti-BCMA/anti-CD3 TCB antibodies (83A10-TCB, 13A4-TCBcv) were analyzedby flow cytometry for binding to human BCMA on BCMA-expressing NCI-H929cells (ATCC® CRL-9068™). MKN45 (human gastric adenocarcinoma cell linethat does not express BCMA) was used as negative control. Briefly,cultured cells were harvested, counted and cell viability was evaluatedusing ViCell. Viable cells were then adjusted to 2×10⁶ cells per ml inBSA-containing FACS Stain Buffer (BD Biosciences). 100 μl of this cellsuspension were further aliquoted per well into a round-bottom 96-wellplate and incubated with 30 μl of the anti-BCMA antibodies orcorresponding IgG control for 30 min at 4° c. All Anti-BCMA/anti-CD3 TCBantibodies (and TCB controls) were titrated and analyzed in finalconcentration range between 1-300 nM. Cells were then centrifuged (5min, 350×g), washed with 120 μl/well FACS Stain Buffer (BD Biosciences),resuspended and incubated for an additional 30 min at 4° C. withfluorochrome-conjugated PE-conjugated AffiniPure F(ab′)2 Fragment goatanti-human IgG Fc Fragment Specific (Jackson Immuno Research Lab;109-116-170). Cells were then washed twice with Stain Buffer (BDBiosciences), fixed using 100 ul BD Fixation buffer per well (#BDBiosciences, 554655) at 4° C. for 20 min, resuspended in 120 μl FACSbuffer and analyzed using BD FACS CantoII. As depicted in FIGS. 25A-25D,the mean fluorescence intensity of anti-BCMA/anti-CD3 TCB antibodieswere plotted in function of antibody concentrations; (A) 83A10-TCB onH929 cells and MKN45 cells, (B) 83A10-TCBcv on H929 cells and MKN45cells. When applicable, EC50 were calculated using Prism GraphPad(LaJolla, Calif., USA) and EC50 values denoting the antibodyconcentration required to reach 50% of the maximal binding for thebinding of 83A10-TCB and 83A10-TCBcv to H929 cells are summarized inTable 13. FIG. 25C shows that 83A10-TCB and 83A10-TCBcv bind to H929cells in a concentration-dependent manner and with similar potency. Suchresults are expected since 83A10-TCB and 83A10-TCBcv molecules shareidentical CDR sequences on the respective VL and VH variable domains.DP47-TCB control antibody did not bind to BCMA-positive H929 myelomacells as measured by a lack of increase in median fluorescenceintensity. In a second head-to-head comparison experiment, 83A10-TCB and83A10-TCBcv were evaluated for binding to BCMA-positive H929 cells andlack of binding to BCMA/CD3-negative MKN45 cells. As depicted in FIG.25D, 83A10-TCB and 83A10-TCBcv bind to BCMA-positive H929 cells in aconcentration-dependent manner and with similar potency. EC50 values forthe binding of 83A10-TCB and 83A10-TCBcv to H929 cells for this secondexperiment are summarized in Table 14.

TABLE 13 EC50 values for binding of anti-BCMA/anti-CD3 TCB antibodies toH929 cells (Experiment 1). Anti-BCMA/ EC50 EC50 anti-CD3 TCB molecules(nM) (μg/ml) 83A10-TCB 9.8 1.9 83A10-TCBcv 14.5 2.8

TABLE 14 EC50 values for binding of anti-BCMA/anti-CD3 TCB antibodies toH929 cells (Experiment 2). Anti-BCMA/ EC50 EC50 anti-CD3 TCB molecules(nM) (μg/ml) 83A10-TCB 16.9 3.25 83A10-TCBcv 14.5 2.8

Redirected T-Cell Cytotoxicity of BCMA-High Expressing H929 MyelomaCells Induced by Anti-BCMA/Anti-CD3 T Cell Bispecific Antibodies (LDHRelease Assay)

Anti-BCMA/anti-CD3 TCB antibodies were also analyzed for their potentialto induce T cell-mediated apoptosis in BCMA-high expressing myelomacells upon crosslinking of the construct via binding of the antigenbinding moieties to BCMA on cells. Briefly, human BCMA-expressing H929multiple myeloma target cells were harvested with Cell DissociationBuffer, washed and resuspended in RPMI supplemented with 10% fetalbovine serum (Invitrogen). Approximately 30,000 cells per well wereplated in a round-bottom 96-well plate and the respective dilution ofthe antibody construct was added for a desired final concentration (intriplicates); final concentrations ranging from 0.1 pM to 10 nM. For anappropriate comparison, all TCB constructs and controls were adjusted tothe same molarity. Human total T cells (effector) were added into thewells to obtain a final effector:target (E:T) ratio of 5:1. When humanPBMC were used as effector cells, a final E:T ratio of 10:1 was used.Negative control groups were represented by effector or target cellsonly. As a positive control for the activation of human pan T cells, 1μg/ml PHA-M (Sigma #L8902) was used. For normalization, maximal lysis ofthe H929 MM target cells (=100%) was determined by incubation of thetarget cells with a final concentration of 1% Triton X-100, inducingcell death. Minimal lysis (=0%) was represented by target cellsco-incubated with effector cells only, i.e. without any T cellbispecific antibody. After 20-24 h incubation at 37° C., 5% CO₂, LDHrelease from the apoptotic/necrotic myeloma target cells into thesupernatant was then measured with the LDH detection kit (Roche AppliedScience), following the manufacturer's instructions. The percentage ofLDH release was plotted against the concentrations of anti-BCMA/anti-CD3T cell bispecific antibodies in concentration-response curves. Whenapplicable, the EC50 values were measured using Prism software(GraphPad) and determined as the TCB antibody concentration that resultsin 50% of maximum LDH release. As shown in FIGS. 26A-26D,anti-BCMA/anti-CD3 TCB antibodies ((A,B) 83A10-TCB, (C,D) 83A10-TCBcv)induced a concentration-dependent killing of BCMA-positive H929 myelomacells as measured by LDH release. The killing of H929 cells was specificsince DP47-TCB control antibody which does not bind to BCMA-positivetarget cells did not induce LDH release, even at the highestconcentration of 1 nM (A). Even though EC50 values were not measurablewith the use of Prism (GraphPad) statistical software for 83A10-TCB (A,B) and 83A10-TCBcv (C, Experiment 1), the magnitude of EC50 values couldbe approximately estimated to low picomolar range potency for bothnon-charged and charged TCB molecules. In a second experiment, theeffect of 83A10-TCBcv was evaluated in the redirected T-cell killingassay and an EC50 value could be measured to 1.5 pM. The authors couldnot exclude that the slightly lower EC50 value (slightly better potency)could be due to blood donor variability. However, the magnitude ofpotency to kill H929 cells was definitely in the low picomolar range.The overall results suggest that 83A10-TCB (without charge variant) vs.83A10-TCBcv (with charge variant) shows similar biological properties incell-based assays.

TABLE 15 EC50 values and estimations for redirected T-cell killing ofH929 cells induced by anti-BCMA/anti-CD3 TCB antibodies. Anti-BCMA/ EC50EC50 anti-CD3 TCB molecules (pM) (μg/ml) 83A10-TCB (Experiment 1) Low pMrange Single digit (approx. <20) 83A10-TCB (Experiment 2) Low pM rangeSingle digit (approx. <20) 83A10-TCBcv (Experiment 1) Low pM rangeSingle digit (approx. <20) 83A10-TCBcv (Experiment 2) 1.5 0.3

Example 3

Preparation of “2+1 IgG CrossFab, Inverted” T-Cell Bispecific Antibodywith Charge Modifications (Anti-Her2/Anti-CD3) and “2+1 IgG CrossFab”T-Cell Bispecific Antibody with Charge Modifications(Anti-Her3/Anti-CD3)

A schematic illustration of the molecules prepared in this example isshown in FIGS. 27A-27B. The anti-Her2/anti-CD3 “2+1 IgG CrossFab,inverted” molecule with charge modifications (referred to in thisexample as “Her2 TCB”) comprises the amino acid sequences of SEQ ID NOs21, 52, 53 and 54. The anti-Her3/anti-CD3 “2+1 IgG CrossFab” moleculewith charge modifications (referred to in this example as “Her3 TCB”)comprises the amino acid sequences of SEQ ID NOs 21, 55, 56 and 57. Themolecules were prepared, purified and analyzed as described in Example 1above (with a single preparative SEC step).

Both molecules could be purified with high final quality shown byanalytical size exclusion chromatography and CE-SDS (Tables 16 and 17).Although recovery of the Her2 TCB in this preparation was lower comparedto the Her3 TCB, the protein was almost pure after the two purificationsteps (Protein A and SEC). CE-SDS analysis shows only 1.18% lowmolecular weight impurity at approximately 164 kDa (Table 17). Thespecies detected at 187.28 kDa corresponds to the target moleculewithout N-linked glycosylation on the Fc domain (this species iscommonly detected by CE-SDS for human IgG₁ after production ineukaryotic cells).

Her3 TCB could be purified with good recovery. The final quality wassuperior to the Her2 TCB comparing the final monomer content. Also theCE-SDS shows 100% target protein, assuming the peak detected at 192.05kDa corresponds to the non-glycosylated Fc-species.

For both preparations no product-related low molecular weight impuritiessuch as free light chains (expected molecular weight at 25 kDa),dimerized light chains as it can occur by introducing only a CH1-CLexchange on one light chain (expected molecular weight at 50 kDa) ormolecules with missing or non-covalently linked light chains (expectedmolecular weight at 125 kDa, 150 kDa or 175 kDa) have been detected byCE-SDS or analytical size exclusion chromatography.

TABLE 16 Summary of production and purification of anti-Her2/anti-CD3and anti-Her3/anti-CD3 TCB molecules with charge modifications.Analytical SEC Titer Recovery Yield (HMW/Monomer/LMW) Molecule [mg/l][%] [mg/l] [%] Her2 TCB 45 1.8 1 3.3/96.7/0 Her3 TCB 72 12.7 11.420/100/0

TABLE 17 CE-SDS analyses (non-reduced) of anti-Her2/anti-CD3 and anti-Her3/anti-CD3 TCB molecules with charge modifications. Molecule Peak #Size [kDa] Purity [%] Her2 TCB 1 163.99 1.18 2 187.28 1.30 3 200.8197.52 Her3 TCB 1 192.05 19.36 2 198.57 80.64

Binding of Her2 TCB and Her3 TCB to Cells

Jurkat suspension cells were harvested, washed with FACS buffer(PBS+0.1% BSA) once and viability was determined by ViCell.

Adherent KPL-4 tumor cells (kindly provided by J. Kurebayashi, KawasakiMedical School, Japan) were harvested with Cell Dissociation Buffer(Gibco Invitrogen) and washed with FACS buffer once, before viabilitywas determined by ViCell.

0.2 million cells were plated per well of a round-bottom 96-well plateand the plates were centrifuged for 4 min at 400 g. Then 25 μl per wellof the TCB dilutions in FACS buffer was added to the cells. The cellswere incubated for 30 min in the fridge. Afterwards the cells werewashed twice with 150 μl FACS buffer per well.

25 μl of appropriately diluted secondary antibody (FITC conjugatedAffiniPure F(ab′)₂ Fragment, Goat Anti-Human IgG, F(ab′)₂ fragmentspecific, Jackson ImmunoResearch) were added per well and the plateswere stained for further 30 min at 4° C. in the dark.

The plates were washed twice with 150 μl FACS buffer per well andresuspended in 150 μl FACS buffer. The analysis was performed using a BDFACS CantoII, equipped with FACS Diva Software. Median fluorescencevalues (MFI) were plotted against the concentration of the TCBmolecules.

As shown in FIGS. 29A-29B, both TCBs show concentration-dependent goodbinding to their respective target antigens on cells.

Activation of Human CD8⁺ T Effector Cells, after T Cell-Mediated Lysisof Human Tumor Cells, Induced by the Her3 TCB

CD8⁺ T effector cells of a classical tumor cell lysis experiment (asdescribed below) with Her3 TCB using an effector-to-target ratio (E:T)of 10:1 and an incubation time of 48h were evaluated for the percentageof CD69-positive cells.

Briefly, after incubation, PBMCs were transferred to a round-bottom96-well plate, centrifuged at 350×g for 5 min and washed twice with PBScontaining 0.1% BSA. Surface staining for CD8 (Biolegend #300908) andCD69 (BioLegend #310904) was performed according to the suppliers'indications. Cells were washed twice with 150 μl/well PBS containing0.1% BSA and fixed for 20 min at 4° C. using 100 μl/well 1% PFA. Aftercentrifugation, the samples were resuspended in 200 μl/well PBS 0.1% BSAand analyzed at FACS CantoII (Software FACS Diva).

As shown in FIG. 30 , the Her3 TCB induces cross-linkage of T cells andtumor cells (KPL-4) via its respective targeting moieties and inducesactivation of T cells in a concentration-dependent manner.

Jurkat-NFAT Activation Assay

The capacity of the Her2 TCB and the Her3 TCB to induce T cellcross-linking and subsequently T cell activation, was assessed usingco-cultures of tumor antigen positive target cells (KPL-4) andJurkat-NFAT reporter cells (a CD3-expressing human acute lymphaticleukemia reporter cell line with a NFAT promoter, GloResponse JurkatNFAT-RE-luc2P, Promega #CS176501). Upon simultaneous binding of the TCBmolecule to human Her2, respectively human Her3, antigen (expressed ontumor cells) and CD3 antigen (expressed on Jurkat-NFAT reporter cells),the NFAT promoter is activated and leads to expression of active fireflyluciferase. The intensity of luminescence signal (obtained upon additionof luciferase substrate) is proportional to the intensity of CD3activation and signaling. For the assay, KPL-4 human tumor cells wereharvested with Cell Dissociation Buffer (Gibco Invitrogen) and viabilitywas determined using ViCell. 20 000 cells/well were plated in aflat-bottom, white-walled 96-well-plate (Greiner bio-one) and dilutedTCBs or medium (for controls) was added. Subsequently, Jurkat-NFATreporter cells were harvested and viability assessed using ViCell. Cellswere resuspended in cell culture medium and added to tumor cells toobtain a final E:T of 2.5:1 (for Her2 TCB) or 5:1 (for Her3 TCB) asindicated, and a final volume of 100 μl per well. Cells were incubatedfor 5 h at 37° C. in a humidified incubator. At the end of theincubation time, 100 μl/well of ONE-Glo solution (Promega, #E6120) (1:1ONE-Glo and assay medium volume per well) were added to wells andincubated for 10 min at room temperature in the dark. Luminescence wasdetected using WALLAC Victor3 ELISA reader (PerkinElmer2030), 5 sec/wellas detection time.

As depicted in FIGS. 31A and 31B, both TCB molecules induce T cellcross-linking via CD3 and subsequently T cell activation. The Her3 TCBis slightly more potent on KPL-4 cells, which might be explained by ahigher level of Her3 over Her2 on these target cells.

Tumor Cell Lysis Induced by Her2 TCB and Her3 TCB

Tumor cell lysis of Her2- or Her3-expressing tumor target cells inducedby the respective TCB molecules was assessed, using human peripheralblood mononuclear cells (PBMCs) as effectors, at an E:T of 10:1. Tumorcell lysis was determined by measurement of released LDH into thesupernatants after 24 h and 48 h upon incubation with the TCBs, asindicated.

Human PBMCs were isolated from fresh blood or from a buffy coat.Briefly, blood was diluted 2:1 (fresh blood) or 3:1 (buffy coat) withPBS. About 30 ml of the blood/PBS mixture was layered on 15 ml ofHistopaque (Sigma) and centrifuged for 30 min at 450×g without brake atRT. The lymphocytes were collected with a 10 ml pipette into 50 ml tubescontaining PBS. The tubes were filled up to 50 ml with PBS andcentrifuged 10 min at 350 g. The supernatant was discarded, the pelletre-suspended in 50 ml PBS and centrifuged for 10 min at 300×g. Thewashing step was repeated once. The cells were re-suspended in RPMIcontaining 10% FCS and 1% GlutaMax (Life Technologies) and stored at 37°C., 5% CO₂ in the incubator until assay start (not longer than 24h).

Target cells were harvested with Trypsin/EDTA, washed, and plated atdensity of 30 000 cells/well using flat-bottom 96-well plates. Cellswere left to adhere overnight in a humidified incubator. On the day ofthe assay, the assay plates were centrifuged at 350×g for 5 min and themedium was aspirated. 100 μl per well of assay medium were added.

The TCBs were added at indicated concentrations (range of 0.001 pM-1 nMfor the Her3 TCB, and 0.01 pM-100 nM for the Her2 TCB, in triplicates).PBMCs were added to target cells at the final E:T ratio of 10:1. Targetcell killing was assessed after 24 h and 48 h of incubation byquantification of LDH (lactate dehydrogenase) released into cellsupernatants by apoptotic/necrotic cells (LDH detection kit, RocheApplied Science, #11 644 793 001). Maximal lysis of the target cells(=100%) was achieved by incubation of target cells with 1% Triton X-100.Minimal lysis (=0%) refers to target cells co-incubated with effectorcells without bispecific antibody. The EC50 values were calculated usingGraphPadPrism5.

In another experiment, tumor cell lysis was determined by Caspase 3/7activity after 6.5h by measuring luminescence in a microplate reader (5s reading time per wells).

For the determination of Caspase 3/7 activity, KPL-4-Caspase-3/7GloSensor target cells (KPL-4 cells stably transfected with GioSensorplasmid) were harvested as described above. After one wash with PBS theconcentration was adjusted to 0.3×10⁶ cells/ml in the assay medium(RPMI1640, 2% FCS, 1% Glutamax) and mixed with 2% v/v GloSensor cAMPReagent (Promega). 100 μl (=30 000 cells) of this target cell suspensionwas transferred into each well of a 96-flat bottom plate with whitewalls. Peripheral blood mononuclear cells (PBMCs) were prepared byHistopaque density centrifugation of enriched lymphocyte preparations(buffy coats) obtained from healthy human donors, as described above.The tumor cell lysis assay was performed essentially as described above.

The results depicted in FIG. 32C and FIG. 33 illustrate that the Her3TCB molecule induces potent and concentration-dependent apoptosis andlysis of KPL-4 tumor cells.

The same is true for the Her2 TCB that is depicted FIGS. 32A and 32B andshows significant, concentration-dependent lysis of tumor cells overtime. Thereby, the EC50 of killing seems to depend on the expressionlevel of Her2 on the respective target cell. The higher the expressionlevel, the better the tumor cell killing by the Her2 TCB.

Example 4

Preparation of “(Fab)₂-CrossFab” T-Cell Bispecific Antibodies with andwithout Charge Modifications (Anti-MCSP/Anti-CD3)

A schematic illustration of the molecules prepared in this example isshown in FIGS. 34A-34B. The anti-MCSP/anti-CD3 “(Fab)₂-CrossFab”molecule with charge modifications in the MCSP binders (referred to as“(Fab)2-XFab-LC007cv” in this example) comprises the amino acidsequences of SEQ ID NOs 58, 59 and 60. The anti-MCSP/anti-CD3“(Fab)₂-CrossFab” molecule without charge modifications (referred to as“(Fab)2-XFab” in this example) comprises the corresponding amino acidsequences without the charge modifications.

The molecules were prepared, purified and analyzed essentially asdescribed in Example 1 above, with the following adaptations.

For the production of these molecules, the HEK293-EBNA cells weretransfected with the corresponding expression vectors in a 1:2:1 ratio(“vector heavy chain”: “vector light chain anti-MSCP Fab”: “vector lightchain anti-CD3 Fab”).

Concentration of the constructs in the culture medium was determined byProteinA-HPLC, based on binding of parts of the CH1 domain to ProteinAat pH8.0 and step elution from pH2.5 as described in Example 1.

The secreted proteins were purified from cell culture supernatants byaffinity chromatography using affinity chromatography binding to CH1,followed by a size exclusion chromatographic step.

For affinity chromatography, supernatant was loaded on a HiTrapKappaSelect column (CV=5 mL, GE Healthcare) equilibrated with 5 ml 50 mMTris, 100 mM glycine, 150 mM NaCl pH 8.0. Unbound protein was removed bywashing with at least 10 column volumes 50 mM Tris, 100 mM glycine, 150mM NaCl pH 8.0. The target protein was eluted in 10 column volumesgradient to 50 mM Tris, 100 mM glycine, 150 mM NaCl pH 2.0. Proteinsolution was neutralized by adding 1/40 of 2 M Tris pH 8.0. Targetprotein is concentrated and filtered prior loading on a HiLoad Superdex200 column (GE Healthcare) equilibrated with 20 mM histidine, 140 mMsodium chloride, 0.01% Tween-20, pH 6.0. Both molecules were producedand purified following the same method Compared to the molecule withoutcharge modifications (“(Fab)₂-XFab”) the titer of the molecule withcharges was 10 fold lower. Nevertheless the final recovery wasapproximately two times higher for the molecule with the chargemodifications in the two anti-MCSP Fabs (“(Fab)2-XFab-LC007cv”) (Table18). The (Fab)2-XFab-LC007cv molecule could be purified to a finalmonomer content of 95.8% shown by size exclusion chromatography and afinal purity proven by CE-SDS analyses of 94.33%.

TABLE 18 Summary of production and purification of anti-MCSP/anti- CD3TCB molecules with and without charge modifications. Analytical SECTiter Recovery Yield (HMW/Monomer/LMW) Molecule [mg/l] [%] [mg/l] [%](Fab)2-XFab 25 6.24 7.8 0/100/0 (Fab)2-XFab- 2.32 10.5 0.24 3.2/95.8/1LC007cv

TABLE 19 CE-SDS analyses (non-reduced) of the anti-MCSP/anti- CD3 TCBmolecule with charge modifications. Molecule Peak # Size [kDa] Purity[%] (Fab)2-XFab- 1 162.67 94.33 LC007cv 2 170.59 5.67

Cell Binding of “(Fab)₂-CrossFab” T-Cell Bispecific Antibodies with andwithout Charge Modifications (Anti-MCSP/Anti-CD3)

Jurkat-NFAT suspension cells were harvested, washed with FACS buffer(PBS+0.1% BSA) once and viability was determined by ViCell.

Adherent MV-3 tumor cells were harvested with Cell Dissociation Buffer(Gibco Invitrogen) and washed with FACS buffer once, before viabilitywas determined by ViCell. 0.2 million cells were plated per well of around-bottom 96-well plate and the plates were centrifuged for 4 min at400×g. Then 25 μl per well of the primary antibody dilutions in FACSbuffer was added to the cells. The cells were incubated for 30 min inthe fridge. Afterwards the cells were washed twice with 150 μl FACSbuffer per well.

25 μl of the diluted secondary antibody (FITC conjugated AffiniPureF(ab′)₂ Fragment, Goat Anti-Human IgG, F(ab′)₂ fragment specific,Jackson ImmunoResearch) were added per well and the plates were stainedfor further 30 min at 4° C. in the dark.

The plates were washed twice with 150 μl FACS buffer per well andresuspended in 150 μl FACS buffer. The analysis was performed using a BDFACS CantoII, equipped with FACS Diva Software. Median fluorescencevalues (MFI) were plotted against the concentration of the MCSP TCBmolecules.

As shown in FIG. 36 , the (Fab)2-XFAb-LC007cv molecule showsconcentration-dependent binding to human MCSP on MV-3 and to human CD3on Jurkat cells. The (Fab)2-XFab molecule without charge modificationsshows comparable binding to human MCSP as (Fab)2-XFAb-LC007cv (EC50binding of 2.3 nM for the (Fab)2-XFAb-LC007cv versus EC 50 1.5 nM forthe (Fab)2-XFab).

Tumor Cell Lysis Mediated by “(Fab)₂-CrossFab” T-Cell BispecificAntibodies with and without Charge Modifications (Anti-MCSP/Anti-CD3)

Tumor cell lysis of MCSP-expressing MV-3 tumor target cells induced bythe MCSP TCB molecules was using human PBMCs as effectors, at an E:T of10:1. Tumor cell lysis was determined by measurement of released LDHinto the supernatants after 24 h and 48 h upon incubation with the TCBs.

Briefly, target cells were harvested with Trypsin/EDTA, washed, andplated at density of 25 000 cells/well using flat-bottom 96-well plates.Cells were left to adhere overnight in a humidified incubator. On theday of the assay, the assay plates were centrifuged at 350×g for 5 minand the medium was aspirated. 100 μl per well of assay medium wereadded.

Peripheral blood mononuclear cells (PBMCs) were isolated from freshblood. Briefly, blood was diluted 2:1 with PBS. About 30 ml of theblood/PBS mixture was layered on 15 ml of Histopaque (Sigma) andcentrifuged for 30 min at 450×g without brake. The lymphocytes werecollected with a 10 ml pipette into 50 ml tubes containing PBS. Thetubes were filled up to 50 ml with PBS and centrifuged 10 min at 350×g.The supernatant was discarded, the pellet re-suspended in 50 ml PBS andcentrifuged for 10 min at 300×g. The washing step was repeated once. Thecells were re-suspended in RPMI containing 10% FCS and 1% GlutaMax (LifeTechnologies) and stored at 37° C., 5% CO₂ in the incubator until assaystart (not longer than 24h).

For the killing assay, the TCB molecules were added at indicatedconcentrations (range of 0.04 pM-10 nM in triplicates). PBMCs were addedto target cells at the final E:T ratio of 10:1. Target cell killing wasassessed after 24 h and 48 h of incubation by quantification of LDH(lactate dehydrogenase) released into cell supernatants byapoptotic/necrotic cells (LDH detection kit, Roche Applied Science, #11644 793 001). Maximal lysis of the target cells (=100%) was achieved byincubation of target cells with 1% Triton X-100. Minimal lysis (=0%)refers to target cells co-incubated with effector cells withoutbispecific antibody. The EC50 values were calculated usingGraphPadPrism5.

As depicted in FIG. 37 , both molecules show concentration-dependentlysis of hMCSP-expressing target cells. The potency of the(Fab)₂-XFAb-LC007cv molecule (EC50 2.8 pM after 24h, and 8.6 pM after48h) is comparable to the potency of the (Fab)₂-XFab molecule withoutcharge modifications (EC50 5.9 pM after 24 h, and 4.8 pM after 48 h).

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, the descriptions and examples should not be construed aslimiting the scope of the invention. The disclosures of all patent andscientific literature cited herein are expressly incorporated in theirentirety by reference.

1. A T cell activating bispecific antigen binding molecule comprising(a) a first Fab molecule which specifically binds to a first antigen (b)a second Fab molecule which specifically binds to a second antigen, andwherein the variable domains VL and VH of the Fab light chain and theFab heavy chain are replaced by each other, wherein the first antigen isan activating T cell antigen and the second antigen is a target cellantigen, or the first antigen is a target cell antigen and the secondantigen is an activating T cell antigen; and wherein i) in the constantdomain CL of the first Fab molecule under a) the amino acid at position124 is substituted independently by lysine (K), arginine (R) orhistidine (H) (numbering according to Kabat), and wherein in theconstant domain CH1 of the first Fab molecule under a) the amino acid atposition 147 or the amino acid at position 213 is substitutedindependently by glutamic acid (E), or aspartic acid (D) (numberingaccording to Kabat EU index); or ii) in the constant domain CL of thesecond Fab molecule under b) the amino acid at position 124 issubstituted independently by lysine (K), arginine (R) or histidine (H)(numbering according to Kabat), and wherein in the constant domain CH1of the second Fab molecule under b) the amino acid at position 147 orthe amino acid at position 213 is substituted independently by glutamicacid (E), or aspartic acid (D) (numbering according to Kabat EU index).2. The T cell activating bispecific antigen binding molecule of claim 1,wherein the first antigen is a target cell antigen and the secondantigen is an activating T cell antigen.
 3. The T cell activatingbispecific antigen binding molecule of claim 1, wherein the activating Tcell antigen is CD3, particularly CD3 epsilon. 4-5. (canceled)
 6. The Tcell activating bispecific antigen binding molecule according to claim1, wherein (a) in the constant domain CL of the first Fab molecule undera) the amino acid at position 124 is substituted independently by lysine(K), arginine (R) or histidine (H) (numbering according to Kabat) andthe amino acid at position 123 is substituted independently by lysine(K), arginine (R) or histidine (H) (numbering according to Kabat), andwherein in the constant domain CH1 of the first Fab molecule under a)the amino acid at position 147 is substituted independently by glutamicacid (E), or aspartic acid (D) (numbering according to Kabat EU index)and the amino acid at position 213 is substituted independently byglutamic acid (E), or aspartic acid (D) (numbering according to Kabat EUindex); or (b) in the constant domain CL of the second Fab moleculeunder b) the amino acid at position 124 is substituted independently bylysine (K), arginine (R) or histidine (H) (numbering according to Kabat)and the amino acid at position 123 is substituted independently bylysine (K), arginine (R) or histidine (H) (numbering according toKabat), and wherein in the constant domain CH1 of the second Fabmolecule under b) the amino acid at position 147 is substitutedindependently by glutamic acid (E), or aspartic acid (D) (numberingaccording to Kabat EU index) and the amino acid at position 213 issubstituted independently by glutamic acid (E), or aspartic acid (D)(numbering according to Kabat EU index). 7-13. (canceled)
 14. The T cellactivating bispecific antigen binding molecule according to claim 1,further comprising: c) a third Fab molecule which specifically binds tothe first antigen and/or is identical to the first Fab molecule; and/ord) an Fc domain composed of a first and a second subunit capable ofstable association. 15-17. (canceled)
 18. The T cell activatingbispecific antigen binding molecule according to claim 1, wherein: (a)the first and the second Fab molecule are fused to each other,optionally via a peptide linker; and/or (b) the second Fab molecule isfused at the C-terminus of the Fab heavy chain to the N-terminus of theFab heavy chain of the first Fab molecule, or the first Fab molecule isfused at the C-terminus of the Fab heavy chain to the N-terminus of theFab heavy chain of the second Fab molecule. 19-20. (canceled)
 21. The Tcell activating bispecific antigen binding molecule according to claim14, wherein (i) the second Fab molecule is fused at the C-terminus ofthe Fab heavy chain to the N-terminus of the first or the second subunitof the Fc domain; (ii) the first Fab molecule is fused at the C-terminusof the Fab heavy chain to the N-terminus of the first or the secondsubunit of the Fc domain; (iii) the first and the second Fab moleculeare each fused at the C-terminus of the Fab heavy chain to theN-terminus of one of the subunits of the Fc domain; and/or (iv) thethird Fab molecule is fused at the C-terminus of the Fab heavy chain tothe N-terminus of the first or second subunit of the Fc domain.
 22. TheT cell activating bispecific antigen binding molecule of claim 14,wherein: (a) the second and the third Fab molecule are each fused at theC-terminus of the Fab heavy chain to the N-terminus of one of thesubunits of the Fc domain, and the first Fab molecule is fused at theC-terminus of the Fab heavy chain to the N-terminus of the Fab heavychain of the second Fab molecule; or (b) the first and the third Fabmolecule are each fused at the C-terminus of the Fab heavy chain to theN-terminus of one of the subunits of the Fc domain, and the second Fabmolecule is fused at the C-terminus of the Fab heavy chain to theN-terminus of the Fab heavy chain of the first Fab molecule. 23.(canceled)
 24. The T cell activating bispecific antigen binding moleculeaccording to claim 22, wherein the first and the third Fab molecule andthe Fc domain are part of an immunoglobulin molecule, particularly anIgG class immunoglobulin. 25-26. (canceled)
 27. The T cell activatingbispecific antigen binding molecule according to claim 1, wherein (a)the activating T cell antigen is CD3, particularly CD3 epsilon, and theFab molecule which specifically binds to the activating T cell antigencomprises the heavy chain complementarity determining region (CDR) 1 ofSEQ ID NO: 4, the heavy chain CDR 2 of SEQ ID NO: 5, the heavy chain CDR3 of SEQ ID NO: 6, the light chain CDR 1 of SEQ ID NO: 8, the lightchain CDR 2 of SEQ ID NO: 9 and the light chain CDR 3 of SEQ ID NO: 10and/or a heavy chain variable region comprising an amino acid sequencethat is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to theamino acid sequence of SEQ ID NO: 3 and a light chain variable regioncomprising an amino acid sequence that is at least about 95%, 96%, 97%,98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 7;and/or (b) the target cell antigen is CD20 and the Fab molecule whichspecifically binds to the target cell antigen comprises the heavy chaincomplementarity determining region (CDR) 1 of SEQ ID NO: 46, the heavychain CDR 2 of SEQ ID NO: 47, the heavy chain CDR 3 of SEQ ID NO: 48,the light chain CDR 1 of SEQ ID NO: 49, the light chain CDR 2 of SEQ IDNO: 50 and the light chain CDR 3 of SEQ ID NO: 51 and/or a heavy chainvariable region comprising an amino acid sequence that is at least about95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence ofSEQ ID NO: 30 and a light chain variable region comprising an amino acidsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO:
 31. 28-30. (canceled)31. A T cell activating bispecific antigen binding molecule comprisinga) a first Fab molecule which specifically binds to a first antigen; b)a second Fab molecule which specifically binds to a second antigen, andwherein the variable domains VL and VH of the Fab light chain and theFab heavy chain are replaced by each other; c) a third Fab moleculewhich specifically binds to the first antigen; and d) an Fc domaincomposed of a first and a second subunit capable of stable association;wherein (i) the first antigen is CD20 and the second antigen is CD3,particularly CD3 epsilon; (ii) the first Fab molecule under a) and thethird Fab molecule under c) each comprise the heavy chaincomplementarity determining region (CDR) 1 of SEQ ID NO: 46, the heavychain CDR 2 of SEQ ID NO: 47, the heavy chain CDR 3 of SEQ ID NO: 48,the light chain CDR 1 of SEQ ID NO: 49, the light chain CDR 2 of SEQ IDNO: 50 and the light chain CDR 3 of SEQ ID NO: 51, and the second Fabmolecule under b) comprises the heavy chain CDR 1 of SEQ ID NO: 4, theheavy chain CDR 2 of SEQ ID NO: 5, the heavy chain CDR 3 of SEQ ID NO:6, the light chain CDR 1 of SEQ ID NO: 8, the light chain CDR 2 of SEQID NO: 9 and the light chain CDR 3 of SEQ ID NO: 10; (iii) in theconstant domain CL of the first Fab molecule under a) and the third Fabmolecule under c) the amino acid at position 124 is substituted bylysine (K) (numbering according to Kabat) and the amino acid at position123 is substituted by lysine (K) or arginine (R), particularly byarginine (R) (numbering according to Kabat), and wherein in the constantdomain CH1 of the first Fab molecule under a) and the third Fab moleculeunder c) the amino acid at position 147 is substituted by glutamic acid(E) (numbering according to Kabat EU index) and the amino acid atposition 213 is substituted by glutamic acid (E) (numbering according toKabat EU index); and (iv) the first Fab molecule under a) is fused atthe C-terminus of the Fab heavy chain to the N-terminus of the Fab heavychain of the second Fab molecule under b), and the second Fab moleculeunder b) and the third Fab molecule under c) are each fused at theC-terminus of the Fab heavy chain to the N-terminus of one of thesubunits of the Fc domain under d).
 32. The T cell activating bispecificantigen binding molecule of claim 31, wherein: (i) the first Fabmolecule under a) and the third Fab molecule under c) each comprise aheavy chain variable region comprising the amino acid sequence of SEQ IDNO: 30 and a light chain variable region comprising the amino acidsequence of SEQ ID NO: 31; and/or (ii) the second Fab molecule under b)comprises a heavy chain variable region comprising the amino acidsequence of SEQ ID NO: 3 and a light chain variable region comprisingthe amino acid sequence of SEQ ID NO:
 7. 33. (canceled)
 34. The T cellactivating bispecific antigen binding molecule according to claim 14,wherein the Fc domain is an IgG, specifically an IgG₁ or IgG₄, Fc domainand/or is a human Fc domain.
 35. (canceled)
 36. The T cell activatingbispecific antigen binding molecule according to claim 14, wherein theFc domain comprises a modification promoting the association of thefirst and the second subunit of the Fc domain.
 37. The T cell activatingbispecific antigen binding molecule of claim 34, wherein in the CH3domain of the first subunit of the Fc domain an amino acid residue isreplaced with an amino acid residue having a larger side chain volumeselected from the group consisting of arginine (R), phenylalanine (F),tyrosine (Y), and tryptophan (W), thereby generating a protuberancewithin the CH3 domain of the first subunit which is positionable in acavity within the CH3 domain of the second subunit, and in the CH3domain of the second subunit of the Fc domain an amino acid residue isreplaced with an amino acid residue having a smaller side chain volumeselected from the group consisting of alanine (A), serine (S), threonine(T), and valine (V), thereby generating a cavity within the CH3 domainof the second subunit within which the protuberance within the CH3domain of the first subunit is positionable.
 38. (canceled)
 39. The Tcell activating bispecific antigen binding molecule of claim 37,wherein: (a) in the CH3 domain of the first subunit of the Fc domain thethreonine residue at position 366 is replaced with a tryptophan residue(T366W), and in the CH3 domain of the second subunit of the Fc domainthe tyrosine residue at position 407 is replaced with a valine residue(Y407V), and optionally in the second subunit of the Fc domainadditionally the threonine residue at position 366 is replaced with aserine residue (T366S) and the leucine residue at position 368 isreplaced with an alanine residue (L368A) (numberings according to KabatEU index); and/or (b) in the first subunit of the Fc domain additionallythe serine residue at position 354 is replaced with a cysteine residue(S354C) or the glutamic acid residue at position 356 is replaced with acysteine residue (E356C), and in the second subunit of the Fc domainadditionally the tyrosine residue at position 349 is replaced by acysteine residue (Y349C) (numberings according to Kabat EU index).40-41. (canceled)
 42. The T cell activating bispecific antigen bindingmolecule according to claim 14, wherein the Fc domain: (a) exhibitsreduced binding affinity to an Fc receptor and/or reduced effectorfunction, as compared to a native IgG₁ Fc domain; (b) comprises one ormore amino acid substitution that reduces binding to an Fc receptorand/or effector function at one or more position selected from the groupof L234, L235, and P329 (Kabat EU index numbering); and/or (c) is an Fcγreceptor. 43-49. (canceled)
 50. A method of producing a T cellactivating bispecific antigen binding molecule capable of specificbinding to CD3 and a target cell antigen, comprising the steps of a)culturing a host cell comprising one or more isolated polynucleotidesencoding the T cell activating bispecific antigen binding molecule ofclaim 1 under conditions suitable for the expression of the T cellactivating bispecific antigen binding molecule and b) recovering the Tcell activating bispecific antigen binding molecule.
 51. (canceled) 52.A pharmaceutical composition comprising the T cell activating bispecificantigen binding molecule of claim 1 and a pharmaceutically acceptablecarrier.
 53. A method of treating a disease in an individual, comprisingadministering to said individual a therapeutically effective amount of acomposition comprising the T cell activating bispecific antigen bindingmolecule of claim 1 in a pharmaceutically acceptable form, wherein thedisease is cancer. 54-55. (canceled)